Labeling reagents and labeled targets comprising nonmetallic porphyrins

ABSTRACT

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

This is a divisional of U.S. patent application Ser. No. 10/096,075,filed on Mar. 12, 2002 now U.S. Pat. No. 7,166,478.

FIELD OF THE INVENTION

This invention relates to the field of labeling chemistry includinglabeling reagents, processes for target labeling, labeled targets,processes for preparing labeling reagents, and the like. This inventionalso relates to the use of such compositions and processes in otherprocesses for nucleic acid and enzymatic activity determinations andanalyses.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to concurrently filed U.S. patentapplication Ser. No. 10/096,076, filed Mar. 12, 2002, Rabbani et al.,that application being titled “Real-Time Nucleic Acid DetectionProcesses and Compositions.” The contents of the aforementioned Ser. No.10/096,076, are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

For purposes of organization, this background has been divided intoseven parts as follows:

(1) Reactive Groups of Labeling Reagents

(2) Linker Arms for Connecting Labels to Targets

(3) Porphyrin Fluorescent Dyes as Labels

(4) Alterations in Fluorescent Properties

(5) Fluorescent Intercalators

(6) Chemiluminescence

(6) Real Time Detection through Fluorescence

(7) Primer Binding Sequences in Analytes

(1) Reactive Groups of Labeling Reagents

The use of non-radioactive labels in biochemistry and molecular biologyhas grown exponentially in recent years. Among the various compoundsused as non-radioactive labels, aromatic dyes that produce fluorescentor luminescent signal are especially useful. Notable examples of suchcompounds include fluorescein, rhodamine, coumarin and cyanine dyes suchas Cy3 and Cy5. Composite dyes have also been synthesized by fusing twodifferent dyes together (Lee et al., (1992) Nucl. Acids Res. 20;2471-2488; Lee et al., U.S. Pat. No. 5,945,526 and Waggoner et al., inU.S. Pat. No. 6,008,373, all of which are hereby incorporated byreference).

Non-radioactive labeling methods were initially developed to attachsignal-generating groups onto proteins. This was achieved by modifyinglabels with chemical groups such that they would be capable of reactingwith the amine, thiol, and hydroxyl groups that are naturally present onproteins. Examples of reactive groups that were used for this purposeincluded activated esters such as N-hydroxysuccinimide esters,isothiocyanates and other compounds. Consequently, when it becamedesirable to label nucleotides and nucleic acids by non-radioactivemeans, methods were developed to convert nucleotides and polynucleotidesinto a form that made them functionally similar to proteins. Forinstance, U.S. Pat. No. 4,711,955 (incorporated by reference) disclosedthe addition of amines to the 8-position of a purine, the 5-position ofa pyrimidine and the 7-position of a deazapurine. The same methods thatcould add a label to the amine group of a protein could now be appliedtowards these modified nucleotides.

Among the compounds used as fluorescent labels, the cyanine-based dyeshave become widely used since they have high extinction coefficients andnarrow emission bands. Furthermore, modifications can be made in theirstructure that can alter the particular wavelengths where thesecompounds will absorb and fluoresce light. The cyanine dyes have thegeneral structure comprising two indolenine based rings connected by aseries of conjugated double bonds. The dyes are classified by the number(n) of central double bonds connecting the two ring structures;monocarbocyanine or trimethinecarbocyanine when n=1; dicarbocyanine orpentamethinecarbocyanine when n=2; and tricarbocyanine orheptamethinecarbocyanine when n=3. The spectral characteristics of thecyanine dyes have been observed to follow specific empirical rules. Forexample, each additional conjugated double bond between the rings willraise the absorption and emission maximum about 100 nm. Thus, when acompound with n=1 has a maximum absorption of approximately 550 nm,equivalent compounds with n=2 and n=3 will have maximum absorptions of650 nm and 750 nm respectively. Addition of aromatic groups to the sidesof the molecules can shift the absorption by 15 nm to a longerwavelength. The groups comprising the indolenine ring can alsocontribute to the absorption and emission characteristics. Using thevalues obtained with gem-dimethyl group as a reference point, oxygensubstituted in the ring for the gem-dimethyl group decreases theabsorption and emission maxima by approximately 50 nm. In contrast,substitution of sulfur increases the absorption and emission maxima byabout 25 nm. R groups on the aromatic rings such as alkyl,alkyl-sulfonate and alkyl-carboxylate have little effect on theabsorption and emission maxima of the cyanine dyes (U.S. Pat. No.6,110,630).

Cyanine dyes synthesized with arms containing functional groups havebeen prepared with iodoacetamide, isothiocyanate and succinimidyl estersthat react with sulfhydryl groups on proteins (Ernst, et al., (1989),Cytometry 10, 3-10; Mujumdar, et al., (1989), Cytometry 10, 11-19;Southwick, et al., (1990) Cytometry 11, 4187-430). A new series ofmodified dyes were prepared which contained a sulfonate group on thephenyl portion of the indolenine ring. (Mujumdar et al., (1993)Bioconjugate Chemistry 4; 105-111 hereby incorporated by reference) thatincreased the water solubility of the dyes. These dyes were activated bytreatment with disuccinimidyl carbonate to form succinimidyl esters thatwere then used to label proteins by substitution at the amine groups.Other activating groups have since been placed on the cyanine dyes. InU.S. Pat. No. 5,627,027 and U.S. Pat. No. 5,268,486 (incorporated byreference), cyanine dyes were prepared which comprise isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, mono or di-halogensubstituted pyridine, mono or di-halogen substituted diazine, aziridine,sulfonyl halide, acid halide, hydroxy-succinimide ester,hydroxy-sulfosuccinimide ester, imido esters, glyoxal groups andaldehydes and other groups, all of which can form a covalent bond withan amine, thiol or hydroxyl group on a target molecule.

In U.S. Pat. No. 6,110,630 (incorporated by reference), cyanine dyeswere prepared with a series of reactive groups derived fromN-hydroxynaphthalimide. These groups included hydroxysuccinimide,para-nitrophenol, N-hydroxyphtalimide and N-hydroxynaphtalimide all ofwhich can react with nucleotides modified with primary amines. The samechemical reactions that have been described above have also been used inU.S. Pat. No. 6,114,350 (incorporated by reference) but with theconstituents reversed. In this disclosure, the cyanine dyes weremodified with amine, sulfhydryl or hydroxyl groups and the targetmolecules were modified to comprise the appropriate reactive groups.

Cyanine dyes containing arms that comprise reactive functional groupshave been prepared by the general scheme in which the entireheterocyclic compound comprising the two indolenine structures and theintervening unsaturated chain was synthesized first; the terminalreactive groups or any other functionality necessary to link the dyes toproteins or nucleic acids were then added after the completion of thewhole dimeric dye unit.

(2) Linker Arms for Connecting Labels to Targets

Labeled nucleotides have been used for the synthesis of DNA and RNAprobes in many enzymatic methods including terminal transferaselabeling, nick translation, random priming, reverse transcription, RNAtranscription and primer extension. Labeled phosphoramidite versions ofthese nucleotides have also been used with automated synthesizers toprepare labeled oligonucleotides. The resulting labeled probes arewidely used in such standard procedures as northern blotting, Southernblotting, in situ hybridization, RNAse protection assays, DNA sequencingreactions, DNA and RNA microarray analysis and chromosome painting.

There is an extensive literature on chemical modification of nucleicacids by means of which a signal moiety is directly or indirectlyattached to a nucleic acid. Primary concerns of this art have been withregard to which site in a nucleic acid is used for attachment i.e.sugar, base or phosphate analogues and whether these sites aredisruptive or non-disruptive (see for instance the disclosures of U.S.Pat. Nos. 4,711,955 and 5,241,060; both patents incorporated byreference), the chemistry at the site of attachment that allows linkageto a reactive group or signaling moiety a spacer group usuallyconsisting of a single aromatic group (U.S. Pat. Nos. 4,952,685 and5,013,831, both hereby incorporated by reference) or a carbon/carbonaliphatic chain to provide distance between the nucleic acid and areactive group or signaling moiety and a reactive group at the end ofthe spacer such as an OH, NH, SH or some other group that can allowcoupling to a signaling moiety and the nature of the signaling moiety.

Although the foregoing have all been descriptions of the various aspectsthat are concerned with the synthesis of modified nucleotides andpolynucleotides, they have also been shown to be significant factorswith regard to the properties of the resultant nucleotides andpolynucleotides. Indeed, there have been numerous demonstrations thatthe modified nucleotides described in the present art have shortcomingscompared to unmodified nucleotides.

For instance, these factors can have major impact on the ability ofthese modified nucleotides to be incorporated by polymerases. Aconsequence of this is that when using a modified base as the solesource of that particular nucleotide, there may be a loss in the amountof nucleic acid synthesis compared to a reaction with unmodifiednucleotides. As a result of this, modified nucleotides are usuallyemployed as part of a mixture of modified and unmodified versions of agiven nucleotide. Although this restores synthesis to levels comparableto reactions without any modified nucleotides, a bias is often seenagainst the use of the modified version of the nucleotide. As such, thefinal proportion of modified/unmodified nucleotide may be much lowerthan the ratio of the reagents. Users then have a choice of either usingnucleic acids that are minimally labeled or of decreased yields. Whencomparable modified nucleotides are used that only comprise a linker armattached to a base (such as allylamine dUTP) difficulties withincorporation are seldom seen. As such, the foregoing problem is likelyto be due to the interactions of the label with either the polymerase orthe active site where synthesis is taking place.

Difficulties in the use of polymerases can be bypassed by the use ofoligonucleotide synthesizers where an ordered chemical joining ofphosphoramidite derivatives of nucleotides can be used to producelabeled nucleic acids of interest. However, the presence of signalagents on modified nucleotides can even be problematic in this system.For instance, a phosphoramidite of a modified nucleotide may display aloss of coupling efficiency as the chain is extended. Although this maybe problematic in itself, multiple and especially successive use ofmodified nucleotides in a sequence for a synthetic oligonucleotide canresult in a drastic cumulative loss of product. Additionally, chemicalsynthesis is in itself not always an appropriate solution. There may becircumstances where labeled nucleic acids need to be of larger lengthsthan is practical for a synthesizer. Also, an intrinsic part ofsynthetic approaches is a necessity for a discrete sequence for thenucleic acid. For many purposes, a pool or library of nucleic acidswould require an impractically large number of different species forsynthetic approaches.

An example of a method to increase the yield of labeled oligonucleotidesor polynucleotide is to use a non-interfering group such as anallylamine modified analogue during synthesis by either a polymerase oran oligonucleotide synthesizer. Labeling is then carried outpost-synthetically by attachment of the desired group through thechemically reactive allylamine moieties. However, in this case, althoughincorporation or coupling efficiency may be restored, there may still beproblems of the coupling efficiencies of attachment of the desired groupto the allylamine. For instance, coupling of labels to allylaminemoieties in a nucleic acid is dramatically less efficient fordouble-stranded DNA compared to single-stranded targets. In addition topotential yield problems, the functionality of the modification may beaffected by how it is attached to a base. For instance if a hapten isattached to a base, the nature of the arm separating the hapten from thebase may affect its accessibility to a potential binding partner. When asignal generating moiety is attached through a base, the nature of thearm may also affect interactions between the signal generating moietyand the nucleotide and polynucleotide.

Attempts to limit these deleterious interactions have been carried outin several ways. For instance, attachment of the arm to the base hasbeen carried out with either a double bond alkene group (U.S. Pat. No.4,711,955) or a triple bond alkyne group (U.S. Pat. No. 5,047,519)thereby inducing a directionality of the linker away from the nucleotideor polynucleotide. However, this approach is of limited utility sincethis rigidity is limited to only the vicinity of the attachment of thelinker to the base. In addition, attempts at limiting interactions havebeen carried out by having the arm displace the active or signal groupaway from the nucleotide or polynucleotide by lengthening the spacergroup. For instance, a commercially available modified nucleotideincluded a seven carbon aliphatic chain (Cat. No. 42724, ENZO Biochem,Inc. New York, N.Y.) between the base and a biotin moiety used forsignal generation. This product was further improved by the substitutionof linkers with 11 or even 16 carbon lengths (Cat. Nos. 42722 and 42723,also available from ENZO Biochem, Inc. New York, N.Y.). A comparison wasalso carried out using different length linker arms and a cyanine dyelabeled nucleotide (Zhu et al., 1994 Nucl. Acid Res. 22; 3418-3422). Adirect improvement in efficiency was noted as the length was increasedfrom 10 to 17 and from 17 to 24. However, even with the longest linker,it could be seen that there was incomplete compensation for the presenceof the fluorescent marker in terms of efficiency. This may be a resultof the fact that due to the flexibility of the aliphatic carbon chainused for this spacer segment, the reporter groups will seldom be foundin a conformation where they are completely extended away from thenucleotide itself. Thus, although this approach changed the length ofthe linker, it was not a change in the flexible nature of the spacer.

In an attempt to circumvent this problem, in U.S. Pat. No. 5,948,648,Khan et al. have disclosed the use of multiple alkyne or aromatic groupsconnecting a marker to a nucleotide. However, this method employs highlynon-polar groups in the linker that may induce interaction between thelinker and the marker, thereby limiting its effectiveness by decreasingcoupling efficiencies or by increasing non-specific binding by labeledcompounds that include these groups. In addition, these groups maydecrease the water solubility of either the labeled compound or variousintermediates used to make the labeled compound.

The continued difficulties in using activated or labeled nucleotideswhich have incorporated the foregoing features demonstrates that thereare still deleterious interactions occurring between the base,oligonucleotide or polynucleotide and the moiety at the end of the armin methods of the previous art. Although the foregoing has beendescribed with respect to attachment to nucleic acids, these problemsare shared with other groups for which it may be useful to attach amarker or label.

(3) Porphyrin Fluorescent Dyes as Labels

Assays that employ fluorescently labeled probes depend upon illuminationat one particular wavelength and detection of the emission at anotherwavelength (the Stokes shift). There exists an extensive literature onthe variety of compounds that have various excitation/emission spectralcharacteristics suitable for such assays. When fluorescent compounds areused for comparative expression analysis, the ability to carry outsignal detection simultaneously for each label depends upon how markedis the difference between the labels. Thus, fluorophores such as Cy 3and Cy 5 are commonly used in expression analysis since they haveemission peaks at 570 and 667 respectively. One class of compounds thathas not been effectively exploited for this analysis are the porphyrins.

The ability of porphyrins to absorb light energy and efficiently releaseit has been used in a number of other systems. For example, lightinduced cleavage of nucleic acids can be carried out by a number ofmetallo-porphyrins that are either free in solution or attached to asequence specific oligonucleotide (Doan et al., (1986). Biochemistry 26;6736-6739). One application of this system has been the targeting andkilling of cancer cells through light induced DNA damage afterabsorption of metallo-phorphyrins (Moan et al., (1986) Photochemistryand Photobiology 43; 681-690). Another example of the high energeticability of metallo-phorphyrins can be seen with their use as catalyticagents (Forgione et al., U.S. Pat. No. 4,375,972) for non-enzymaticchemiluminescence. Futhermore, there are cases where phorphyrins havebeen used as labeling reagents, for example Roelant et al in U.S. Pat.No. 6,001,573 and Hendrix in U.S. Pat. No. 5,464,741 (herebyincorporated by reference) where Pd octaethylporphyrins were convertedto the isothiocyanate and used as labeling reagents particularly for usein immunoassays. However, in these cases metallic phorphyrins wereexclusively used.

The drawback of the use of metallo-phorphyrins is that the destructiveabilities of these compounds are counter-productive when used in arrayanalysis or other assay systems which require the maintenance of theintegrity of the nucleic acid strands of analytes or probes. Therefore,it would be highly advantageous to be able to utilize porphyrins fortheir fluorescent and chemiluminescent properties while eliminatingtheir nucleic acid destructive properties.

(4) Alterations in Fluorescent Properties

In previous art, it has been shown that the addition of phenylacetylenegroups to anthracene increases the emission maxima 72 nm. (Maulding andRoberts, 1968 J Org Chem). Furthermore, the Stokes shift, the differencebetween the absorption and emission maxima, was also increased by theaddition of the phenyl acetylene group to the anthracene dye.Specifically the difference of 6 nm was increased to 31 nm following theaddition of two phenyl acetylene groups. When the phenyl acetylene groupwas added to naphthacene the difference between the absorption andemission maxima increased from 7 nm to 32 nm. Furthermore, the quantumyields of anthracene and naphtacene was significantly increased by theaddition of the phenyl acetylene groups to them.

The application of this effect was limited to these compounds becausethe chemistries and reactions used for the addition of thesesubstituents required ketone or aldehyde groups. Also, addition ofunsaturated groups to dyes has the undesired effect of potentiallydecreasing their solubility in aqueous solutions. In addition, themodified anthracene dyes described by Maulding and Roberts lacked anyreactive groups that could be used for attachment.

(5) Fluorescent Intercalators

Intercalating dyes have been used for the detection and visualization ofDNA in many techniques including the detection of DNA in electrophoresisgels, in situ hybridization, flow cytometry and real time detection ofamplification. An intercalating dye with a long history of popular usageis ethidium bromide. Ethidium bromide has the useful properties of highaffinity for nucleic acids and an increased fluorescence after binding.This enhancement of fluorescence takes place with both single-strandedand double-stranded nucleic acids with the double-stranded DNA showing amuch more marked effect, generally around thirty-fold. Other dyes whichexhibit increased fluorescence signal upon binding to nucleic acid havebeen developed in recent years including such compounds as acridineorange, SYBR Green and Picogreen. There is continually a need, however,for increased signal generation after the binding or intercalation withnucleic acids especially for the use in techniques, such as real timeamplification.

(6) Chemiluminescence

The use of chemiluminescent reagents for signal detection has gainedwider use in recent years. There are several different classes ofcompounds that can produce luminescent signals including 1,2-dioxetanesand luminols. 1,2-Dioxetanes are four-membered rings which contain twoadjacent oxygens. Some forms of these compounds are very unstable andemit light as they decompose. On the other hand, the presence of anadamantyl group can lead to a highly stable form with a half-life ofseveral years (Wieringa et al. (1972) Tetrahedron Letters 169-172incorporated by reference). Use can be made of this property by using astable form of a 1,2-dioxetane as a substrate in an enzyme linked assaywhere the presence of the enzyme will transform the substrate into anunstable form thereby using chemiluminescence for signal generation.Enzymatic induction of a chemiluminescent signal has been describedwhere an adamantyl dioxetane derivative was synthesized with anadditional group that was a substrate for enzymatic cleavage (U.S. Pat.No. 5,707,559, Schaap et al. (1987) Tetrahedron Letters, 28; 935-938;Schaap et al. (1987) Tetrahedron Letters, 28; 1159-1163, all of whichare incorporated by reference). In the presence of the appropriateenzyme, cleavage would take place and an unstable compound would beformed that emitted light as it decomposes.

A common design of dioxetane derivatives for this method is attachmentof an aryl group that has hydroxyl substituents which contain protectinggroups. The removal of the protecting group by the appropriate enzymeresults in a negatively charged oxygen. This intermediate is unstableand leads to the decomposition of the compound and the emission oflight. Various 1,2-dioxetane derivatives have been developed that can beactivated by different enzymes depending upon the nature of theprotecting group. Enzymes that have been described as potentially usefulfor this purpose have included alkaline phosphatase, galactosidase,glucosidase, esterase, trypsin, lipase, and phospholipase among others(for instance, see U.S. Pat. No. 4,978,614, incorporated herein byreference).

Variations of this basic method have also been disclosed. For example,Urdea has disclosed (U.S. Pat. No. 5,132,204, incorporated by reference)stable 1,2-dioxetanes derivatives which require the activity of twoenzymes in order to produce a signal. Haces has disclosed a method wherethe decomposition of the 1,2-dioxetane is triggered by an enzymatic orchemical reaction which releases a terminal nucleophile (U.S. Pat. No.5,248,618 incorporated by reference). This can now undergo anintramolecular substitution reaction, thereby liberating a phenoxy groupwhich triggers the decomposition of the 1,2-dioxetane. The chain wherethe intramolecular reaction takes place is made up of single bonds thusallowing complete rotational freedom around all the bonds and relying ona random interaction between the groups participating in theintramolecular reaction.

Despite improvements within the field of chemiluminescent signalingthere still exists the need for new substrates and reagents. Many of thesubstrates that are currently available produce a high level ofbackground due to enzyme independent triggering of the decomposition ofthe substrate and release of chemiluminescent signal. Therefore, a newtype of 1,2-dioxetane which is more stable in the absence of an enzymewould be a desirable reagent.

(7) Real Time Detection through Fluorescence

Amplification of nucleic acids from clinical samples has become a widelyused technique. The first methodology for this process, the PolymeraseChain Reaction (PCR), was described by Mullis et al. in U.S. Pat. No.4,683,202 hereby incorporated by reference. Since that time, othermethodologies such as Ligation Chain Reaction (LCR) (U.S. Pat. No.5,494,810), GAP-LCR (U.S. Pat. No. 6,004,286), Nucleic Acid SequenceBased Amplification (NASBA) (U.S. Pat. No. 5,130,238), StrandDisplacement Amplification (SDA) (U.S. Pat. Nos. 5,270,184 and5,455,166) and Loop Mediated Amplification (U.S. patent application Ser.No. 09/104,067; European Patent Application Publication No. EP 0 971 039A) have been described, all of which are incorporated by reference.Detection of an amplified product derived from the appropriate targethas been carried out in number of ways. In the initial method describedby Mullis et al., gel analysis was used to detect the presence of adiscrete nucleic acid species. Identification of this species as beingindicative of the presence of the intended target was determined by sizeassessment and the use of negative controls lacking the target sequence.The placement of the primers used for amplification dictated a specificsize for the product from appropriate target sequence. Spuriousamplification products made from non-target sequences were unlikely tohave the same size product as the target derived sequence.Alternatively, more elaborate methods have been used to examine theparticular nature of the sequences that are present in the amplificationproduct. For instance, restriction enzyme digestion has been used todetermine the presence, absence or spatial location of specificsequences. The presence of the appropriate sequences has also beenestablished by hybridization experiments. In this method, theamplification product can be used as either the target or as a probe.

The foregoing detection methods have historically been used after theamplification reaction was completed. More recently, methods have beendescribed for measuring the extent of synthesis during the course ofamplification, i.e. “real-time” detection. For instance, in the simplestsystem, an intercalating agent is present during the amplificationreaction (Higuchi in U.S. Pat. No. 5,994,056 and Wittwer et al., U.S.Pat. No. 6,174,670; both of which are hereby incorporated by reference).This method takes advantage of an enhancement of fluorescence exhibitedby the binding of an intercalator to double-stranded nucleic acids.Measurement of the amount of fluorescence can take placepost-synthetically in a fluorometer after the reaction is over, or realtime measurements can be carried out during the course of the reactionby using a special PCR cycler machine that is equipped with afluorescence detection system and uses capillary tubes for the reactions(U.S. Pat. Nos. 5,455,175 and 6,174,670 hereby incorporated byreference). As the amount of double-stranded material rises during thecourse of amplification, the amount of signal also increases. Thesensitivity of this system depends upon a sufficient amount ofdouble-stranded nucleic acid being produced that generates a signal thatis distinguishable from the fluorescence of a) unbound intercalator andb) intercalator molecules bound to single-stranded primers in thereaction mix. Specificity is derived from the nature of theamplification reaction itself or by looking at a Tm profile of thereaction products. Although the initial work was done with EthidiumBromide, SYBR Green™ is more commonly used at the present time. Avariation of this system has been described by Singer and Haugland inU.S. Pat. No. 6,323,337 B1 (incorporated by reference), where theprimers used in PCR reactions were modified with quenchers therebyreducing signal generation of a fluorecent intercalator that was boundto a primer dimer molecule. Signal generation from target derivedamplicons could still take place since amplicons derived from targetsequences comprised intercalators bound to segments that weresufficiently distant from the quenchers.

Another method of analysis that depends upon incorporation has beendescribed by Nazarenko (U.S. Pat. No. 5,866,336; incorporated byreference). In this system, signal generation is dependent upon theincorporation of primers into double-stranded amplification products.The primers are designed such that they have extra sequences added ontotheir 5′ ends. In the absence of amplification, stem-loop structures areformed through intramolecular hybridization that consequently bring aquencher into proximity with an energy donor thereby preventingfluorescence. However, when a primer becomes incorporated intodouble-stranded amplicons, the quencher and donor become physicallyseparated and the donor is now able to produce a fluorescent signal. Thespecificity of this system depends upon the specificity of theamplification reaction itself. Since the stem-loop sequences are derivedfrom extra sequences, the Tm profile of signal generation is the samewhether the amplicons were derived from the appropriate target moleculesor from non-target sequences.

In addition to incorporation based assays, probe based systems have alsobeen used for real-time analysis. For instance, a dual probe system canbe used in a homogeneous assay to detect the presence of appropriatetarget sequences. In this method, one probe comprises an energy donorand the other probe comprises an energy acceptor (European PatentApplication Publication No. 0 070 685 by Michael Heller, published Jan.26, 1983). Thus, when the target sequence is present, the two probes canbind to adjacent sequences and allow energy transfer to take place. Inthe absence of target sequences, the probes remain unbound and no energytransfer takes place. Even if by chance, there are non-target sequencesin a sample that are sufficiently homologous that binding of one or bothprobes takes place, no signal is generated since energy transfer wouldrequire that both probes bind and that they be in a particular proximityto each other. Advantage of this system has been taken by Wittwer etal., in U.S. Pat. No. 6,174,670 (incorporated by reference) for realtime detection of PCR amplification using the capillary tube equippedPCR machine described previously. The primer annealing step during eachindividual cycle can also allow the simultaneous binding of each probeto target sequences providing an assessment of the presence and amountof the target sequences. In a further refinement of this method, one ofthe primers comprises an energy transfer element and a single energytransfer probe is used. Labeled probes have also been used inconjunction with fluorescent intercalators to allow the specificity ofthe probe methodology to be combined with the enhancement offluorescence derived from binding to nucleic acids. This was firstdescribed in U.S. Pat. No. 4,868,103 and later applied to amplificationreactions in PCT Int. Appl. WO 99/28500 (both documents incorporated byreference).

Probes have also been used that comprise an energy donor and an energyacceptor in the same nucleic acid. In these assays, the energy acceptor“quenches” fluorescent energy emission in the absence of appropriatecomplementary targets. In one system described by Lizardi et al. in U.S.Pat. No. 5,118,801, “molecular beacons” are used where the energy donorand the quencher are kept in proximity by secondary structures withinternal base pairing. When the target sequences are present,complementary sequences in the Molecular Beacons allow hybridizationevents that destroy this secondary structure thereby allowing energyemission. In another system that has been termed Taqman, use is made ofthe double-stranded selectivity of the exonuclease activity of Taqpolymerase (Gelfand et al., U.S. Pat. No. 5,210,015). When targetmolecules are present, hybridization of the probe to complementarysequences converts the single-stranded probe into a substrate for theexonuclease. Degradation of the probe separates the donor from thequencher thereby releasing light.

(8) Primer Binding Sequences in Analytes

One of the characteristics of eucaryotic mRNA is the presence of poly Atails at their 3′ ends. This particular feature has provided a majoradvantage in working with mRNA since the poly A segment can be used as auniversal primer binding site for synthesis of cDNA copies of anyeucaryotic mRNA. However, this has also led to a certain bias in RNAstudies, since the 3′ ends of mRNA are easily obtained and thoroughlystudied but the 5′ ends lack such consensus sequences. Thus, a largenumber of systems have been described whose major purpose has been togenerate clones that have complete representation of the original 5′ endsequences. This has also been carried over in array analysis forcomparative transcription studies. Since substantially all systems usedfor this purpose are initiated by oligo T priming at the 3′ end of mRNA,sequences downstream are dependent upon the continuation of synthesisaway from the 3′ starting point. However, it is well known that there isan attenuation effect of polymerization as polymerases frequently falloff of templates after synthesis of a particular number of bases.Another effect is generated by the presence of RNase H that is acomponent of most reverse transcriptases. Paused DNA strands may allowdigestion of the RNA near the 3′ end of the DNA thereby separating theuncopied portion of the RNA template from the growing DNA strand. Thiseffect may also occur randomly during the course of cDNA synthesis. Assuch, representation of sequences is inversely proportional to theirdistance from the 3′ poly A primer site.

Although prior art has capitalized extensively on poly A segments ofRNA, it should be recognized that poly A mRNA represents only a portionof nucleic acids in biological systems. Another constraint in prior artis that the use of poly A tails is only available in eucaryotic mRNA.Two areas of especial interest are unable to enjoy this benefit. Onearea is bacterial mRNA since they intrinsically lack poly A additions.The second are is heterologous RNA in eucaryotic systems. For anyparticular eucaryotic gene, there is a considerable amount of geneticinformation that is present in heterologous RNA that is lost by the useof polyadenylated mature forms of transcripts that comprise only exoninformation.

The lack of primer consensus sequence in these systems has necessitatedthe use of alternatives to oligo T priming. In prior art, bacterialexpression studies have been carried out by random priming with octamers(Sellinger et al., 2000 Nature Biotechnology 18; 1262-1268), a selectedset of 37 7-mers and 8-mers (Talaat et al., 2000 Nature Biotechnology18; 679-682) and a set of 4,290 gene specific primers (Tao et al., 1999J. Bact. 181; 6425-6490). The use of large sets of primers asrepresented by random primers and set of gene specific primers requireshigh amounts of primers to drive the reaction and should exhibit poorkinetics due to the sequence complexity of the primers and targets. I.e.for any given sequence in an analyte, there is only a very minuteportion of the primers that are complementary to that sequence. Largesets of random primers also have the capacity to use each other asprimers and templates thereby generating nonsense nucleic acids anddecreasing the effective amounts of primers available. Attempts toimprove the kinetics of priming by increasing the amounts of randomoligonucleotides is very limited. First off, there are physicalconstraints in the amount of oligonucleotides that are soluble in areaction mixture. Secondly, increases in the amount of primers isself-limiting since increased primer concentrations results in increasedself-priming, thereby generating more nonsense sequences and absorptionof reagents that would otherwise be used for analyte dependentsynthesis. Lower concentrations can theoretically be used by decreasingthe complexity (i.e. sequence length) of the primers, but restraints arethen imposed upon the stability of hybrid formation. On the other hand,the discrete sub-set of 7-mers and 8-mers described above requiresknowledge of the complete genome of the intended target organism. Assuch, these will only be used with completely sequenced organisms, and aunique set has to be individually developed for each target organismthus limiting its application. Consensus sequences can be enzymaticallyadded by RNA ligation or poly A polymerase but both of these are slowinefficient processes. Thus there exists a need for methods andcompositions that can efficiently provide stable priming of a largenumber of non-polyadenylated templates of variable or even unknownsequence while maintaining a low level of complexity.

Methods have also been described for the introduction of sequences intoanalytes for the purpose of amplification. For instance,oligonucleotides have also been described that comprise a segmentcomplementary to a target sequence and a segment comprising a promotersequence where the target is either a selected discrete sequence or anatural poly A sequence (U.S. Pat. Nos. 5,554,516 and 6,338,954 (bothpatents incorporated by reference)). After hybridization to a targetmRNA, RNAse H is used to cleave a segment of the analyte hybridized tothe complementary segment and then extend the 3′ end of the analyteusing the promoter segment as a template. Since the oligonucletotidethat is used for these methods has a homogeneous nature, this particularmethod relies upon the extension reaction being initiated before theendonuclease reaction completes digestion of the complementary segmentof the analyte.

SUMMARY OF THE INVENTION

The present invention provides a labeling reagent for labeling a target,the labeling reagent comprising a marker moiety M and a reactive group RM-Rwherein the marker moiety M and the reactive group R are covalentlylinked together, the M comprising at least one moiety that comprises aligand, a dye, or both a ligand and a dye; and the reactive group Rbeing capable of forming a carbon-carbon linkage with the target.

The present invention also provides a process for labeling a target, theprocess comprising the steps of (a) providing: (i) the target; (ii) alabeling reagent comprising a marker moiety M and a reactive group RM-Rwherein the marker moiety M and the reactive group R are covalentlylinked together, the M comprising at least one moiety that comprises aligand, a dye, or both a ligand and a dye, and the reactive group Rbeing capable of forming a carbon-carbon linkage with the target; and(b) reacting the target (i) and the labeling reagent (ii) underconditions such that a carbon-carbon linkage forms between the target(i) and the labeling reagent (ii), thereby labeling the target (i) withthe marker moiety M.

This invention also provides a labeled target, the target having beenlabeled by a process comprising the steps of (a) providing: (i) thetarget; (ii) a labeling reagent comprising a marker moiety M and areactive group RM-Rwherein the marker moiety M and the reactive group R are covalentlylinked together, the M comprising at least one moiety that comprises aligand, a dye, or both a ligand and a dye; and the reactive group Rbeing capable of forming a carbon-carbon linkage with the target; (b)reacting the target (i) and the labeling reagent (ii) under conditionssuch that a carbon-carbon linkage forms between the target (i) and thelabeling reagent (ii), thereby labeling the target (i) with the markermoiety M.

Also provided by this invention is a process for preparing a cyanine dyelabeling reagent, the process comprising the steps of (a) providing:

-   -   (i) a first intermediate compound comprising:

wherein X₁ comprises carbon, oxygen, nitrogen or sulfur; and

-   -   (ii) a second intermediate compound comprising:

wherein X₁ comprises carbon, oxygen, nitrogen or sulfur; wherein atleast one of R₁ through R₁₀ comprises a reactive group capable offorming a carbon-carbon linkage with a target, and (ii) linking reagentssuitable for linking the first intermediate compound and the secondintermediate compound; (b) forming a reaction mixture comprising thefirst intermediate compound (i), the second intermediate compound (ii),and the linking reagents under conditions to link (i) and (ii) to form

wherein at least one of R₁ through R₁₀ comprises a reactive groupcapable of forming a carbon-carbon linkage with a target, and wherein nis an integer of 1, 2 or 3, and wherein X₁ and X₂ independently comprisecarbon, oxygen, nitrogen or sulfur.

Further provided by this invention is a labeling reagent comprising anaphenylic analog of a rhodamine dye, the analog comprising at least onereactive group for attaching the labeling reagent to a target, the atleast one reactive group being attached directly to the analog orindirectly through a linker arm.

The present invention also concerns a labeled nucleotide comprising anaphenylic analog of a rhodamine dye, wherein the dye is attacheddirectly to the nucleotide or indirectly through a linker.

This invention also provides a labeled target comprisingT--L--Mwherein T is a target, M is a marker moiety and L is a chemical groupcovalently linking the M to T, the chemical group L comprising abackbone that comprises at least one rigid group that comprises one ormore of:

(d) multimers of (a), (b) or (c), and (e) any combinations of (a), (b),(c) and (d).

The present invention also provides a labeling reagent comprisingR--L--Mwherein R is a reactive group, M is a marker moiety and L is a chemicalgroup covalently linking the M to R, the chemical group L comprising abackbone that comprises at least one rigid group that comprises one ormore of:

(d) multimers of (a), (b) or (c), and (e) any combinations of (a), (b),(c) and (d).

Also provided is a labeled target comprisingT--L--Mwherein T is a target, M is a marker moiety and L is a chemical groupcovalently linking the M to T, the chemical group L comprising abackbone that comprises at least two consecutive polar rigid units.

Additionally provided is a labeling reagent comprisingR--L--Mwherein R is a reactive group, M is a marker moiety and L is a chemicalgroup covalently linking the M to R, the chemical group L comprising abackbone that comprises at least two consecutive polar rigid units.

The invention herein also provides a labeled target comprisingT--L--Mwherein T is a target, M is a marker moiety and L is a chemical groupcovalently linking the M to T, the chemical group L comprising abackbone that comprises at least two consecutive peptide bonds.

Another aspect of this invention is a labeling reagent comprisingR--L--Mwherein R is a reactive group, M is a marker moiety and L is a chemicalgroup covalently linking the M to R, the chemical group L comprising abackbone that comprises at least two consecutive peptide bonds.

Another aspect concerns a labeling reagent comprising a nonmetallicporphyrin, the reagent comprising:

wherein R₀ is a reactive group and is attached directly or indirectly tothe nonmetallic porphyrin, and R₁ through R₈ independently comprisehydrogen, aliphatic, unsaturated aliphatic, cyclic, heterocyclic,aromatic, heteroaromatic, charged or polar groups, or any combinationsof the foregoing.

Further described and provided is a labeled target comprising anonmetallic porphyrin, the reagent comprising:

wherein T is a target molecule attached directly or indirectly to thenonmetallic porphyrin and R₁ through R₈ independently comprise hydrogen,aliphatic, unsaturated aliphatic, cyclic, heterocyclic, aromatic,heteroaromatic, charged or polar groups, or any combinations of theforegoing.

Another part of the present invention is a process for determining theamount of a nucleic acid in a sample of interest, the process comprisingthe steps of: (a) providing: (i) the sample of interest; (ii) a dyecomprising a first phenanthridinium moiety linked to a secondphenanthridinium moiety through the phenyl group in each of the firstand second phenanthridinium moieties; (iii) reagents for carrying outdye binding, hybridization, strand extension, or any combinationthereof; (b) forming a mixture of (i), (ii) and (iii) above, to producea complex comprising the dye (ii) and any nucleic acid that may bepresent in the sample of interest (i); (c) illuminating the mixtureformed in step (b) at a wavelength below 400 nanometers (nm); and (d)measuring fluorescent emission from the illuminated mixture in step (c),the emission being proportional to the quantity of any nucleic acidpresent in the sample of interest (i).

The present invention also provides a composition comprising at leastone of the following dye structures:

Also provided by the present invention is the use of the just-describedcompositions in a process for determining the amount of a nucleic acidin a sample of interest, the process comprising the steps of: (a)providing: (i) the sample of interest; (ii) the dye (a), (b), (c) or (d)from the composition just described; (iii) reagents for carrying out dyebinding, hybridization, strand extension, or any combination thereof;(b) forming a mixture of (i), (ii) and (iii) above, to produce a complexcomprising the dye (ii) and any nucleic acid that may be present in thesample of interest (i); (c) illuminating the mixture formed in step (b)at a first wavelength; and (d) measuring at a second wavelength anyfluorescent emission from the illuminated mixture in step (c), theemission being proportional to the quantity of any nucleic acid presentin the sample of interest (i).

This invention provides a heterodimeric dye composition, the compositioncomprising a first dye that comprises a phenanthridinium moiety; and asecond dye that is different from the first dye, the second dye beingattached through the phenyl ring of the phenanthridium moiety.

This invention also provides a process for determining the amount of anucleic acid in a sample of interest using the last-describedcomposition. The process comprises the steps of: (a) providing: (i) thesample of interest; (ii) the dye last-described; (iii) reagents forcarrying out dye binding, hybridization, strand extension, or anycombination thereof; (b) forming a mixture of (i), (ii) and (iii) above,to produce a complex comprising the dye (ii) and any nucleic acid thatmay be present in the sample of interest (i); (c) illuminating themixture formed in step (b) at a first wavelength; and (d) measuring at asecond wavelength any fluorescent emission from the illuminated mixturein step (c), the emission being proportional to the quantity of anynucleic acid present in the sample of interest (i).

A chemiluminescent reagent is also provided by this invention, thechemiluminescent reagent having the structure:

wherein Q comprises a cycloalkyl or polycycloalkyl group attachedcovalently to the 4-membered ring portion of the dioxetane abovedirectly or indirectly through a linkage group; wherein Z compriseshydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl or cycloheteroalkyl; and wherein R₁ and R₂ comprise chemicalmoieties attached to different sites of a cyclic ring attached to thedioxetane, and wherein R₁ is enzymatically converted into R₁* whichcomprises a chemical reactive group G₁, and wherein R₂ is attached tothe cyclic ring through an oxygen, nitrogen or sulfur atom and comprisesa chemical reactive group G₂ which reacts with the G₁ to convert thedioxetane to an unstable light-emitting dioxetane form.

Using the last-described composition, the invention further provides aprocess for detecting the presence or quantity of enzymatic activity ofinterest in a sample. The process comprises the steps of: (a) providing:(i) the sample suspected of containing enzymatic activity; (ii) thechemiluminescent reagent last-described; (ii) reagents and buffers forcarrying out chemiluminescent reactions; (b) forming a mixture of: (1)(i), (ii) and (iii); or (2) (ii) and (iii) and contacting the mixture of(ii) and (iii) with (i); (c) enzymatically converting thechemiluminescent reagent just described (ii) into an unstablelight-emitting dioxetane form; and (d) measuring the quantity of lightgenerated by the enzymatic conversion in step (c).

Another chemiluminescent reagent provided by the present invention isone having the structure:

wherein Q comprises a cycloalkyl or polycycloalkyl group attachedcovalently to the 4-membered ring portion of the dioxetane abovedirectly or indirectly through a linkage group; wherein Z compriseshydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl or cycloheteroalkyl; and wherein R comprises a chemicallinker having a reactive site attached to the aromatic ring in thestructure; and wherein R′ comprises a substrate for an non-cleavingenzymatic process, wherein the product of the enzymatic process leads tofurther chemical rearrangements that generate an unstable light emittingdioxetane form.

The invention also provides a process for detecting the presence orquantity of enzymatic activity of interest in a sample using thelast-described chemiluminescent reagent. The process comprises the stepsof: (a) providing: (i) the sample suspected of containing enzymaticactivity; (ii) the chemiluminescent reagent last-described; (ii)reagents and buffers for carrying out chemiluminescent reactions; (b)forming a mixture of: (1) (i), (ii) and (iii); or (2) (ii) and (iii) andcontacting the mixture of (ii) and (iii) with (i); (c) enzymaticallyconverting the chemiluminescent reagent just described above (ii) intoan unstable light-emitting dioxetane form; and (d) measuring thequantity of light generated by the enzymatic conversion in step (c).

A dye composition is also provided by this invention, the dyecomposition having the formulaR-Fluorescent Dyewherein R is covalently linked to the Fluorescent Dye comprises two ormore members in combination from a) unsaturated aliphatic groups; b)unsaturated heterocyclic groups; c) aromatic groups; and wherein R iscapable of providing a conjugated system or an electron delocalizedsystem with the fluorescent dye.

A labeled target is further provided, the labeled target having thestructure

wherein the Dye is a fluorescent dye, wherein R is covalently linked tothe Dye, and wherein R comprises two or more members in combination froma) unsaturated aliphatic groups; b) unsaturated heterocyclic groups; c)aromatic groups; and wherein R is capable of providing a conjugatedsystem or an electron delocalized system with the Dye.

Other embodiments and aspects of the present invention are furtherdescribed below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates several examples of linker arms made with rigid polarunits.

FIG. 2 shows structures of various homodimers of Ethidium Bromide: A)meta-EthD B) EthD-1 and C) EthD-2.

FIG. 3 illustrates the use of primers with first and second energytransfer elements:

-   -   double stranded nucleic acid made from extension of two primers    -   double stranded SDA amplicon    -   Nested PCR    -   Ligase Chain Reaction.

FIG. 4 depicts variations of placement of primers in a double strandednucleic acid.

FIG. 5 is an illustration of the use of nucleotides with energy transferelements.

FIG. 6 shows the use of matrix with energy transfer elements.

FIG. 7 is the spectrum of aphenylic analog of TAMRA.

FIG. 8 is the spectrum of aphenylic analog of Texas Red.

FIG. 9 is an outline of method for synthesizing a homodimer.

FIG. 10 depicts the results of illuminating meta-EtBr at 493 nm in thepresence and absence of DNA.

FIG. 11 shows the results of Illuminating meta-EtBr at 350 nm in thepresence and absence of DNA.

FIG. 12 shows the sequence of an HIV antisense amplicon and sequences oftwo primers and one probe used in the examples below to illustrate thenovel use of energy transfer in the present invention.

FIG. 13 shows the use of a CNAC to eliminate a portion of a poly A tailfollowed by incorporation of an oligo C primer binding sequence.

FIG. 14 shows various steps for the synthesis of a dioxetane derivative.

FIG. 15 shows the enzymatic production of an unstable light emittingform of a dioxetane.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel methods and compositions for thepreparation of compounds labeled with ligands and dyes. Included withinthe present disclosure are novel labeling reagents, novel dyes and novelmethods that can be used to synthesize the novel reagents of the presentinvention. The novel methods of the present invention may also beapplied to the synthesis of compounds that have been describedpreviously.

1. Labeling Reagents which Participate in Carbon-Carbon Bond Formation

One aspect of the present invention discloses novel labeling reagentsthat comprise a reactive group capable of creating a carbon-carbon bondbetween a marker or label and a desirable target molecule. This is incontrast to labeling reagents described in prior art which employedprotein derived chemistries involving formation of a bond between anamine, sulfhydryl or hydroxyl group and an appropriate reactive group.The novel labeling reagent of the present invention should provide ahighly efficient means of attaching signal moieties to desirable targetmolecules. Thus, the novel labeling reagents of the present inventioncomprise a ligand or dye portion and a reactive group capable ofcreating a carbon-carbon bond. In addition, it may be desirable toinsert a linker arm that separates the ligand or dye portion from thereactive group. This may provide more efficient coupling between thenovel labeling reagent and an intended target molecule. The presence andnature of the linker arm may also increase the biological or chemicalactivity of the labeled target molecule. The novel reagents of thepresent invention can be used to label any target molecule that iscapable of participating in bond formation with the reactive group ofthe labeling reagent. The target molecule may be in its native state orit may have been modified to participate in formation of a carbon-carbonbond with the novel labeling reagent.

Ligands that may find use with the present invention can include but notbe limited to sugars, lectins, antigens, intercalators, chelators,biotin, digoxygenin and combinations thereof. The particular choice of adye used to synthesize a novel labeling reagent of the present inventionmay depend upon physical characteristics such as absorption maxima,emission maxima, quantum yields, chemical stability and solventsolubility. A large number of fluorescent and chemiluminescent compoundshave been shown to be useful for labeling proteins and nucleic acids.Examples of compounds that may be used as the dye portion can includebut not be limited to xanthene, anthracene, cyanine, porphyrin andcoumarin dyes. Examples of xanthene dyes that may find use with thepresent invention can include but not be limited to fluorescein,6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-Fam), 5- or6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET), 5- or6-carboxy-4′5′2′4′5′7′ hexachlorofluorescein (HEX), 5′ or6′-carboxy-4′,5′-dichloro-2,′7′-dimethoxyfluorescein (JOE),5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE) rhodol, rhodamine,tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine(DTAMRA), rhodamine X (ROX) and Texas Red. Examples of cyanine dyes thatmay find use with the present invention can include but not be limitedto Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5. Other dyes that may finduse with the present invention can include but not be limited to energytransfer dyes, composite dyes and other aromatic compounds that givefluorescent signals. Chemiluminescent compounds that may be used in thepresent invention can include but not be limited to dioxetane andacridinium esters it should also be understood that ligands and dyes arenot mutually exclusive groups. For instance, fluorescein is a well knownexample of a moiety that has been used as a fluorescent label and alsoas an antigen for labeled antibodies.

The reactive group of the novel labeling reagents of the presentinvention is chosen from chemical moieties that are known to be able toparticipate in carbon-carbon bond formation thereby allowing the novellabeling reagent to attach a label to a suitable target molecule.Examples of such reactive groups can comprise but not be limited toalkenes, alkynes, metallo-organic compounds and halogenated compounds.The metallo-organic and halogenated compounds can comprise aromatic,heterocyclic, alkene, and alkyne groups as well as various combinationsthereof. Although such groups have been described previously forsynthesis of labeled compounds, these reactive groups were only used inthe context of adding amino groups to nucleic acids in order to makenucleotides and polynucleotides look like proteins (U.S. Pat. Nos.4,711,955 and 5,047,519, both of which are incorporated by reference).In the present invention, the reactive group of the novel labelingreagent can be attached directly to a ligand or dye, at the terminal endof a linking arm or at an internal site within a linking arm. A reviewof various methods for use of metallo-organic and halogenated compoundsis given by Larock (1982, Tetrahedron Report 128; 1713-1754), Robins etal; (J. Org Chem 1983, 48; 1854-1862), Hobbs and Cocuzza (U.S. Pat. No.5,047,519), Eglinton and McCrae (1963, Advances in Organic Synthesis 4;225-328) and Rieke (2000, Aldrichimica Acta 33; 52-60) all of which areincorporated by reference.

A linking arm that comprises a portion of the novel labeling reagentscan be of any desired length and can be comprised of any suitable atomsthat can include but not be limited to carbon, nitrogen, oxygen, sulfurand any combination thereof. Chemical groups that can comprise thelinker arm can include but not be limited to aliphatic bonds, doublebonds, triple bonds, peptide bonds, aromatic rings, aliphatic rings,heterocyclic rings, ethers, esters, amides, and thioamides. The linkingarm can form a rigid structure or be flexible in nature.

The present invention may be used to label a large variety of targetmolecules. The targets may intrinsically comprise chemical moieties thatcan participate in formation of a carbon-carbon bond with the reactivegroup of the novel labeling reagent or the targets may be modified suchthat they comprise such a group. Examples of chemical moieties on targetmolecules that can combine with the reactive group of the novel labelingreagent can comprise but not be limited to alkenes, alkynes,metallo-organic compounds and halogenated compounds. The metallo-organicand halogenated compounds can comprise aromatic, heterocyclic, alkeneand alkyne groups as well as various combinations thereof. Targetmolecules that may find use with the present invention can include butnot be limited to nucleotides, oligonucleotides, polynucleotides,peptides, oligopeptides, proteins, ligands, synthetic compounds,synthetic polymers, saccharides, polysaccharides, lipids and hormones.Nucleotides that can be labeled by these compounds can include but notbe limited to monophosphates, diphosphates or triphospates. They may beribonucleotides or deoxynucleotides. Modified nucleotides or Nucleotidesanalogues of any of the foregoing may also be used if desired. Examplesof modified nucleotides can include but not be limited to dideoxynucleotides and nucleotides with 3′ amino or 3′ phosphate groups.Examples of nucleotide analogues can include but not be limited topeptide nucleic acids, arabinosides, and acyclo versions. Theseanalogues may be used as nucleotides or as components ofoligonucleotides or polynucleotides. Synthesis of a labeledoligonucleotide or polynucleotide can be carried out by the use ofnucleotides that have been labeled by the novel labeling reagent.Alternatively, modified nucleotides that have chemical groups that canbe used for carbon-carbon bond formation with the novel labelingreagents can be used to synthesize oligonucleotides or polynucleotides.In this method, the presence of reactive groups in the oligonucleotideor polynucleotide products allows a subsequent reaction with the novellabeling reagents of the present invention. Additionally, unmodifiedoligonucleotides and polynucleotides can be chemically treated such thatthey comprise groups capable of participating in carbon-carbon bondformation.

Attachment of the novel labeling reagents of the present invention todesirable target molecules can be carried out by any of a variety ofmeans known to those skilled in the art. For instance, theacetoxymercuration reaction is a well known and established procedurefor introducing covalently bound mercury atoms onto the 5-position ofthe pyrimidine ring or the C-7 position of a deazapurine ring (Dale etal., (1975) Biochemistry 14, 2447-2457, Dale et. al. (1973) Proc. Natl.Acad. Sci. USA 70; 2238-2242. The nucleotides are treated with themercuric acetate in sodium acetate buffer to convert them into mercuricsalts. In the presence of K₂PdCl₄, the addition of a labeled reagent ofthe present invention that has been prepared with a terminal double bondwill allow a carbon-carbon double bond to be formed between the aromaticring of the nucleotide and the terminal carbon of the double bond of thelabeling reagent thereby attaching the label to the nucleotide. In thecase of novel labeling reagents of cyanine dyes with double bonds at theteminus of a linker, the mercuric nucleotide reacts with the double bondat the terminus of the linker arm rather that the aromatic ring or theconjugated double bond between the two rings of the cyanine dye moiety.

In an alternative use of the reaction described above, a novel labelingreagent of the present invention can be prepared where the reactivegroup is a mercury salt. This compound can now react with an unsaturatedbond on the target that is desired to be labeled. This bond may be anintrinsic part of the target molecule or the target molecule may bemodified to include such a group. Reactions can also be carried outwhere both the labeling reagent and the target molecules comprisemercury salts. For instance, Larock (1982 op. cit. Eqns. 146-151) hasdescribed how two groups that each have the structure R1-C═C—HgCl can bejoined together in the presence of appropriate catalysts.

One advantage of the mercuration and palladium catalyzed additionreactions is that they can be carried out in water with a small amountof organic solvent added to increase solubility if necessary. Thisprocedure can be carried out with the nucleotides in any form, forexample with ribonucleotides, deoxynucleotides, dideoxynucleotides andany analogue, as well as with oligonucleotides, oligonucleotideanalogues, protein nucleic acid complexes and polynucleotides.Alternatively the novel labeling reagent can be prepared with a reactivearm containing a terminal triple bond or any other substance which iscapable of forming the carbon-carbon double bond with the targetmolecule.

2. Labeling Proteins by Carbon-Carbon Bond Formation

An important use for the novel labeling reagent may also be forattaching signal groups to proteins. In this particular case,modifications of the protein can be made to make them resemble thenucleic acid target described above. For instance, a target protein canbe reacted with mercuric acetate thereby forming mercurate compounds attyrosine, tryptophan or phenylalanine residues in the protein. Theprotein is now available for reacting with a novel labeling reagent thathas an double bond reactive group where displacement of the mercury willtake place while attaching the label. If desired, thiol groups in theprotein can be protected on the protein by treatment with 2,2′-dipyridyldisulfide prior to the mercuration step.

Amino acids that have primary amines are also sites on a protein thatmay be used with the novel labeling reagent. For instance, proteins thatlack tyrosine groups can be modified with Bolton-Hunter active ester tointroduce tyrosine groups onto primary amines. These can then besubsequently used as described above. Alternatively, the protein can bemodified with acrylic acid active ester to introduce terminal doublebonds into residues that contain primary amines. This modification wouldallow proteins to be used with novel labeling reagents of the presentinvention that have mercurate compounds as reactive groups.

3. Dye Precursors with Reactive Groups for Carbon-Carbon Bond Formation

Attachment of a group to a marker that is suitable for participating ina carbon-carbon bond can be carried out by modification of the marker.On the other hand, attachment can take place with an intermediate thatis used to synthesize a particular marker. For example, cyanine dyelabeling reagents have the following structure:

where n=1, 2 or 3; X1 and X2 can be S, O, N, CH₂ or C(CH₃)₂ and R1-R8comprises a reactive group that could be used to join the cyanine dye toa desirable target molecule. Cyanine dyes are prepared by linkingtogether two indolenine precursor units with an intervening unsaturatedchain. The particular number of units making up the chain determines theparticular absorption and emission spectra of the cyanine dye.

According to the method of the present invention, cyanine dyes can beprepared by attaching a linker arm containing a reactive group capableof generating a carbon-carbon bond to the indolenine ring that is aprecursor to a cyanine dye. This modified indolenine is a novel compoundthat can then be used as a reagent in a reaction where it is coupled toa second indolenine ring through an intervening unsaturated alkyl chainto synthesize a cyanine dye with the structure described above. Thesecond indolenine can be the same as the first or it may be anunmodified version that lacks the linker arm and reactive group. Thesame novel indolenine compound can be used to make a variety ofdifferent cyanine dyes depending upon the nature of the secondindolenine ring and the particular unsaturated chain joining the twoindolenine rings. As a result of this procedure, when the cyanine dyeproduct is formed by joining the precursor rings, it already comprises alinker arm with a reactive group and is ready to be attached to asuitable target molecule.

4. Novel Rhodamine Dyes without the Phenyl Group

In another aspect of the present invention, new dyes and means for theirsynthesis are disclosed. In previous art, derivatives of rhodaminetypically have an aromatic group between the dye and the reactive groupthat is used to attach the rhodamine to a desirable molecule. In thepresent invention, it is disclosed that stable nucleotides can besynthesized that comprise rhodamine analogues where the linker armjoining the dye to a base on the nucleotides lacks the aromatic groupthat is normally present in rhodamine. It is a surprising consequencethat incorporation of such nucleotides becomes more acceptable to apolymerase such that the modified nucleotide can be used without mixingit with unmodified nucleotides. As such, the present invention disclosesthe following novel rhodamine analogues:

where R is a reactive group.5. Rigid Linker Arms

In another aspect of the present invention, methods and compositions aredisclosed that enable modified nucleotides to be used more efficientlyin enzymatic and chemical means of synthesis and/or allow them tofunction more efficiently when they are part of a polynucleotide. In oneembodiment of the present invention, an increased and directedseparation is achieved between a target molecule and the group that hasbeen added to provide a marker or label. In the present invention, thefollowing novel compositions are disclosed that have the formulas:T----L----M and R----L----MIn the diagram above, T is a target for attachment of a marker or label;R is a reactive group that may be used for attachment to a target and Mis a marker or label.

In one aspect of the present invention, L is a chemical group thatcovalently connects the M moiety to the T moiety or R moiety andcomprises one or more of the following groups:

The alkene groups can be in either cis or trans configuration withregard to each other and they may comprise only hydrogen atoms bonded tothe carbon atoms or they may be substituted. In a preferred mode,directionality is derived from having one of the groups aboveimmediately linked to the target, separated by no more than a singleintervening atom or linked to the target through a rigid polar unit.

In another aspect of the present invention, L is a chemical group thatcovalently connects the M moiety to the T moiety and comprises at leasttwo consecutive rigid polar units.

Examples of targets that may find use in the present invention caninclude but not be limited to nucleotides, oligonucleotides,polynucleotides. peptides, polypeptides, cytokines, ligands, haptens,antigens and solid supports. Examples of solid support that may find usewith the present invention can include but not be limited to beads,tubes, plastic slides, glass slides, microchip arrays, wells anddepressions.

Examples of reactive groups that may find use with the present inventioncan include but not be limited to isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono or di-halogen substitutedpyridine, mono or di-halogen substituted diazine, aziridine, sulfonylhalide, acid halide, hydroxy-succinimide ester, hydroxy-sulfosuccinimideester, imido esters, glyoxal groups, aldehydes amines, sulfhydrylgroups, hydroxyl groups. Also included are groups that can participatein carbon carbon bond formation as disclosed previously.

In the present invention, a rigid unit is defined as a group of atomswhere the spatial relations between the atoms are relatively static.

In the present invention, when two moieties are described asconsecutive, the moieties are adjacent or directly next to each other.Additionally, two consecutive moieties can be separated by no more thanone atom, i.e. a single atom.

In the present invention, a rigid unit is non-polar when it essentiallycomprises the same type of atoms. Examples of non-polar rigid unitswould be alkenes, alkynes, unsaturated rings, partially saturated ringsand completely saturated rings that comprise only carbon and hydrogen.

In the present invention, a rigid unit is polar when it comprises atleast two or more different atoms thereby distributing the chargeunequally through the unit. Examples of an arrangement that couldcontribute to polarity could include but not be limited to a carbon atomthat is bonded to N, S, O, P, or a halogen. The heteroatoms that arebonded to the carbon may be used alone or they may be part of polar orcharged functional groups. Examples of the latter can include but not belimited to —OH, —SH, —SO₃, —PO₄, —COOH and —NH₂ groups. The rigid unitscan comprise backbones that are linear, branched or in ring form. Thering forms that also comprise polar or charged functional groupsattached to the rings may be unsaturated rings, partially saturatedrings and completely saturated rings. Multimers of two or more suchpolar rigid units will provide rigid extended arms that create a definedspatial relationship between a target molecule and a marker or signalgenerating moiety.

In the present invention, unsubstituted heterocyclic aromatic compoundswould be considered to be non-polar rigid units due to the electronsharing in the ring. On the other hand, substituted heterocyclicaromatic compounds that comprise polar or charged functional groupsattached to the rings would be considered to be polar rigid units.

Examples of linear polar rigid units that would be useful in the presentinvention can include but not be limited to moieties comprising peptidebonds. Examples of cyclic polar units that have inherent rigidity caninclude but not be limited to sugars. Examples of groups that would haveutility in the present invention that have rigidity derived from theinteractions between subunits can include but not be limited to chargedcomponents where charge repulsions can maximize distances betweensubunits. When negatively charged components are used, there can also berepulsion away from the negatively charged polynucleotide itself. Thelinker may also be designed with bulky side groups that interfere withrotational changes thereby maintaining a discrete spatial structure withregard to the relationship of a base and a signal or reactive group.

The distance of the reactive group or signal moiety from the targetmolecule would be determined by the number and nature of the rigid unitsmaking up the spacer. Thus, a series of three rigid units that comprisean alkene bond followed by two peptide bonds would extend the signalgroup directly away from the nucleotide as shown in FIG. 1A. Thisparticular example would comprise a non-polar rigid unit as well as twopolar rigid units. A series of multiple peptide bonds could stillprovide rigidity while extending the dye or marker further away from thetarget molecule as shown in FIG. 1B. In this particular illustration auracil nucleoside is used as a target and glycine subunits are used toprovide a series of peptide bonds. Different amino acids may also havebeen used if so desired, where the various constituents of the R groupsof the amino acids may be chosen to endow other properties such assolubility or charge upon the rigid arm.

A linker that is comprised of rigid units will depend upon theparticular relationship between the rigid units for whether the overallstructure is rigid or not. For instance, multiple peptide bonds havebeen used in prior art. However, the beneficial qualities of having suchbonds were lost by the inclusion of aliphatic carbon groups in betweenthe peptide. In essence, these were rigid units joined by flexiblelinkers. As seen in FIG. 1, the present invention allows for at most asingle atom between the rigid units, thereby limiting the extent offlexibility between rigid units. Similarly other groups of a non-carbonnature could be used between groups that would retain an overallrigidity while contributing a potentially desirable directionality. Anillustrative example of such a group would be a —S— bond between tworigid units.

As described above, sugar groups may also be used in carrying out thepresent invention. There are a wide variety of sugars that can be usedas individual rigid units and a large number of ways that these sugarscan be linked together either enzymatically or chemically has beenextensively described in the literature.

Although the present invention makes use of two or more polar rigidunits to create a rigid linker arm, it is understood that flexiblegroups and non-polar rigid units may also be included in the rigidlinker arms. For instance, FIG. 1 makes use of an alkene bond betweenthe peptide bonds and the uracil moiety. In addition, additionalflexible units, rigid units or combinations thereof may be includedbetween the last peptide bond and the dye molecule in FIG. 1 whileretaining the effectiveness of the linker.

In the present invention, the presence of such an extended linkage awayfrom a nucleotide should decrease deleterious effects upon incorporationsince the problematic group should be spatially displaced from theactive site where enzymatic incorporation is taking place. In addition,after a modified nucleotide is incorporated either enzymatically orsynthetically, functionality may also be increased by the use of thepresent invention. For instance, extension of a hapten or a chemicallyreactive group further from an oligonucleotide or polynucleotide shouldprovide increased accessibility thereby improving binding or couplingefficiencies. In addition, signal generation groups could also bedisplaced away from the oligonucleotide or polynucleotide by the use ofthe present invention if interference effects are caused by proximity.

The particular point of attachment of the linkers described in thepresent invention may take advantage of previously described art forflexible linkers. As such, the nucleotides may be normal nucleotides orthey may be modified nucleotides or nucleotide analogues with varioussubstituents either added or replacing components in the base, sugar orphosphate moieties as disclosed in U.S. Pat. Nos. 4,711,955; 5,241,060;4,952,685 and 5,013,831; all of which are hereby incorporated byreference). In addition, these modifications may be non-disruptive,semi-disruptive or disruptive. The point of attachment may be the base,sugar or phosphate as described in the previously recited disclosures,but attachment to the base is particularly useful in the presentinvention.

A further benefit of the present invention is that some of the linkersthat have been described may offer beneficial results due to theirchemistry as well as structure. For instance, the last peptide in alinker composed of peptide subunits offers an amine group that may beused to attach useful groups such as signal moieties. In prior art,amine groups were located at the ends of aliphatic chains, with pKvalues of about 11. However, since coupling reactions are usuallycarried out at around pH 8 values, very little of the amine group is ina reactive form at any given time, thereby limiting the efficiency andkinetics of the reaction. In contrast, the amine group at the end ofpeptide chain has a pK of about 9, a value that is more compatible withthe intended coupling reaction. Thereby, the present invention allowsmore effective coupling of a nucleotide or polynucleotide to anappropriate group.

Also, although this particular aspect of the present invention has beendescribed in terms of a rigid linker intervening between a nucleotideand a dye, other applications may also enjoy the benefits of the presentinvention. For instance, labeling of proteins can be improved by usingthe rigid arm of the present invention between the protein and a signalmoiety. Examples of proteins that might enjoy these benefits can includebut not be limited to antibodies, enzymes, cytokines and libraries ofoligopeptides or polypeptides. As described previously for nucleicacids, the use of the present invention may improve the properties ofthe labeled compound as well as the efficiency of the labeling itself.Additionally, there are many procedures that involve fixation of aligand, hapten, protein or a nucleic acid to a solid support. Examplesof such supports can include but not be limited to beads, tubes,microtitre plates, glass slides, plastic slides, microchip arrays, wellsand depressions. The present invention can be used to generate adirected separation of a ligand, hapten, protein or nucleic acid awayfrom the surface of the support. Examples of proteins that might enjoythese benefits can include but not be limited to antibodies, enzymes,cytokines and libraries of oligopeptides or polypeptides.

6. Non-Metallic Porphyrins with Reactive Groups on Non-Pyrrole Positions

In another aspect of the present invention, a novel labeling reagent isdisclosed that comprises a non-metallic porphyrin with a reactive groupat a non-pyrrole position. The spectral quality of non-metallicalkylated porphyrins as fluorescent dyes has been described in Hendrixin U.S. Pat. No. 4,707,454 (incorporated by reference) where Stokesshifts over 150 nm were disclosed. However, when describing reactivegroups for the porphyrins, the only teachings that were disclosed madeuse of chemical groups on the pyrrole positions. Therefore, it is asubject of the present invention that non-metallic porphyrins can bederived which independently comprise hydrogen, aliphatic, unsaturatedaliphatic, cyclic, heterocyclic, aromatic, heteroaromatic, charged orpolar groups on any or all of the eight pyrrole positions and use one ofthe non-pyrrole positions as a site for attaching a reactive group. Thiscomposition has the following structure:

where R_(o) comprises a reactive group attached directly or indirectlyto a non-pyrrole position of the porphyrin (i.e., the α, β, γ, or δpositions) and R₁ through R₈ are as defined just above.

Any of the reactive groups that have been described previously may finduse in the present invention as R₀. R₁ through R₈ may comprise the samegroups or they may be different. If desired, the alkyl groups may alsofurther comprise polar or charged groups that may aid in increasing theaquaeous solubility of the porphyrin. Also if desired, there may be alinker used to attach a reactive group to the porphyrin. The particularrigid arm used in this aspect of the present invention can be any linkerarm that has been previously disclosed or described but it is especiallypreferred that the rigid linker of the present invention be used. Anitro group can be added to a non-pyrrole position as described byFuhrhop and Smith “Laboratory Methods” Chapter 19 in Porphyrins andMetalloporphyrins, Kevin M. Smith, editor, Elsevier ScientificPublishing Company, Amsterdam, 1975, hereby incorporated by reference.Reduction of this group to an amine is well known to those skilled inthe art and further reaction can be carried out to add a linker or areactive group by standard techniques.

Any of the previously described targets may be labeled by thenon-metallic porphyrins of the present invention. For example, thenon-metallic phorphyrins of the present invention may find use byincorporation of a porphryin labeled nucleotide or synthetically by aporphyrin labeled phosphoramidite. Alternatively, oligonucleotide orpolynucleotides can be synthesized that have derivatived nucleotidesthat are suitable for reaction with a chemically compatible derivativeof a non-metallic porphyrin in a post-synthetic step. Labeledoligonucleotides and polynucleotides that comprise the non-metallicporphyrin of the present invention should enjoy a large Stokes shiftwith high efficiency emission. This composition and method of detectionwill enjoy a high level of sensitivity as well as enabling high level ofdiscrimination from other compounds that may be excited at the samewavelength. For instance, if a library of transcripts is labeled withfluorescein and a second library is labeled with octaethylporphine,illumination can be carried out by a single wavelength of 490 nm. Yet,discrimination between the particular fluorophores is easilydistinguishable since the emission peak is 530 nm for fluorescein andthe emission peak for octaethylporphine is 620 nm. At the same time, thequantum yield for the octaethylporphine is comparable to that offluorescein. It is also understood that the non-metallic phorphyrins maybe used in conjunction with any of the other novel methods that aredisclosed herein.

7. Modification of Dyes by Groups that Participate in the Conjugationand/or Electron Delocalized System

In another embodiment of the present invention, methods are disclosedfor the synthesis of novel compositions that comprise two or moreunsaturated compounds added to a fluorescent dye without a requirementfor the presence of kenone groups in an intermediate. In the presentinvention, these unsaturated compounds can be unsaturated aliphaticgroups, unsaturated cyclic compounds, unsaturated heterocycliccompounds, aromatic groups or any combinations thereof. Attachment ofsuch groups allows them to participate in the conjugation and/or theelectron delocalized system (Maulding and Roberts, op. cit. incorporatedby reference) of the dye and confer changes upon the spectralcharacteristics of the dye. These changes can include changing the widthof the excitation and emission peaks, shifting the positions of theexcitation and emission peaks and increasing the quantum yield.

Since addition of unsaturated groups to the dyes may decrease solubilityor result in non-specific hydrophobic interactions, it is an objectiveof the present invention that this effect can be compensated by afurther addition of charged or polar groups. These may be attached tothe dye or to the unsaturated compounds. Also, since these novel dyesfind use as labeling reagents, they may also comprise reactive groupssuitable for attaching the label to desirable target molecules. Thereactive groups can be directly or indirectly linked to the dye, to theunsaturated compounds or to the charged or polar modification groups.

The novel composition of this aspect of the present invention has thefollowing structure:R-Dyewhere Dye is a fluorescent dye; R is covalently linked to the Dye and Rcomprises two or more unsaturated compounds which can be unsaturatedaliphatic groups, unsaturated cyclic compounds, unsaturated heterocycliccompounds, aromatic groups or combinations thereof. Furthermore, one ormore members of R participates in the conjugation and/or electrondelocalized system of the Dye. The unsaturated compounds can besubstituted or unsubstituted. The unsaturated aliphatic group cancomprise an alkene or an alkyne. The aromatic group can comprise aphenyl group, an aryl group, or an aromatic heterocycle. When the groupsare substituted, the substituents can include but not be limited toalkyl groups, aryl groups, alkoxy groups, phenoxy groups, hydroxylgroups, amines, amino groups, amido groups, carboxyl groups, sulfonates,sulfhydryl groups, nitro groups, phosphates or any group that canimprove the properties of the dyes. In the case of an aromatic group, itmay also be substituted by being part of a fused ring structure.Examples of such fused rings can include but not be limited tonaphthalene, anthracene, and phenanthrene.

Groups that can be used as all or part of R can also be described asfollows:

In the diagram above, Ar is an unsaturated cyclic compound, anunsaturated heterocyclic compound or an aromatic group. As describedpreviously, the groups above may be substituted or unsubstituted.

Fluorescent dyes that may find use with the present invention caninclude but not be limited to anthracene, xanthene, cyanine, porphyrin,coumarin and composite dyes. In the case where anthracene is used as theDye with an alkyne joined to the center ring and a phenyl group attachedto the alkyne, the phenyl group will be substituted.

In another aspect of the present invention, a novel composition has thefollowing structure:

where R and Dye are as described previously and where R₁ is covalentlyjoined to R, Dye or to both R and Dye. R₁ further comprises one or morecharged or polar groups to provide additional solubility. This mayuseful when the dye or the dye with the R modification has limitedaquaeous solubility or problems with non-specific hydrophobicinteractions.

In another aspect of the present invention, a novel composition has thestructure

where R, Dye and R₁ are as described previously and where R₂ iscovalently attached to R, Dye, R₁ or any combination thereof and whereR₂ further comprises a reactive group that can be used to attach the dyeto a suitable target molecule. R₂ can comprise any of the reactivegroups previously described including sulfhydryl, hydroxyl and aminegroups, groups capable of reacting with sulfhydryl, hydroxyl and aminegroups, and groups capable of forming a carbon-carbon bond. R₂ canfurther comprise a linker arm that that separates the reactive groupfrom the dye. The linker arm can be of any desirable length and cancomprise a backbone of carbon as well as non-carbon atoms. Non-carbonatioms that may find use can include but not be limited to sulfur,oxygen and nitrogen. The linker arm can comprise saturated, unsaturatedor aromatic groups and may also comprise the rigid arms describedpreviously.

In another aspect of the present invention, a novel labeled targetcomprises the structure:

where R and Dye are as described previously. Targets that may find usewith the present invention can include but not be limited to a protein,a peptide, a nucleic acid, a nucleotide or nucleotide analog, areceptor, a natural or synthetic drug, a synthetic oligomer, a syntheticpolymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, anantigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- orpolysaccharide. The labeled target can also further comprise R₁covalently joined to R, Dye or to both R and Dye. R₁ further comprisesone or more charged or polar groups to provide additional solubility.This may useful when the labeled target or an intermediate used formaking the labeled target has limited aquaeous solubility or problemswith non-specific hydrophobic interactions. The labeled target can alsofurther comprise a linker arm as described above separating the dye fromthe target.8. Intercalators

In another aspect of the present invention, a novel method is disclosedthat provides enhanced discrimination between an intercalating dye thatis bound to a target compared to dye that remains unbound. As describedpreviously, ethidium bromide has been a popular reagent for detectionand visualization of DNA in a number of formats. To investigate theeffect of a second-phenanthridinium ring system on affinity to nucleicacids, a homodimeric form of ethidium bromide was synthesized and tested(Kuhlmann et al., (1978) Nucleic Acids Research, 5; 2629-2633). Thiscompound, N,N-Bis[3-(3,8-diamino-5-methylphenanthridinium-6-yl)benzoyl]1,5-diaminopentane dichloride (meta-EthD) exhibited a muchhigher affinity to nucleic acids than the monomeric form. However, whenfluorescence was measured at the standard wavelength of 493 nm, theincrease in fluorescent emission after binding to nucleic acids wasessentially the same as seen earlier for the ethidium bromide monomer.

It was a surprising and unexpected result that when meta-EthD was usedin a different manner than the standard format, a greatly enhanceddiscrimination between bound and unbound was observed. The presentinvention discloses that when two ethidium bromide molecules are joinedtogether through their phenyl groups, excitation at a wavelength below400 nm can result in an increase of over 150 fold in fluorescentemission upon the binding of DNA to the homodimer as opposed to the 6fold increase seen when the samples are excited at 493 nm.

Two other homodimeric ethidium bromide compounds (EthD-1 and EthD-2) arecommercially available from Molecular Probes, Inc. (Eugene, Oreg.).However, in contrast to the results with meta-EthD,—the discriminationbetween bound and unbound dye was not substantially changed by excitingat wavelengths below 400 nm. It should be pointed out that althoughmeta-EthD, EthD-1 and EthD-2 are all ethidium bromide dimers, they arechemically dissimilar. As shown in (FIG. 2), meta-EthD is comprised oftwo phenanthridinium rings linked together through the meta position ofthe phenyl rings through amide bonds. In contrast, the phenanthridiniumrings of EthD-1 and EthD-2 dimers are joined together through thenitrogen of the center rings rather than through the phenyl rings. Theintervening chain is comprised of an alkyl chain with two amineattachment groups which are secondary in EthD-1 and methylated to givethe quaternary salts for EthD-2. The inability of the EthD-1 and EthD-2compounds to exhibit the same results seen with meta-EthD demonstratesthat the method of the present invention was not a predictable propertyof ethidium dimers per se.

The method of the present invention may find use in many methods thathad been previously described for ethidium bromide, ethidium bromidehomodimers and other intercalators. Of especial use, is the applicationof the present method towards real time analysis of nucleic acidamplification and probes labeled with meta-EthD. The large increase influorescence after illumination at wavelengths under 400 nm will allow abetter signal to noise ratio than previous methods. Thereby, the presentinvention should enjoy a higher sensitivity of detection of thesynthesis of nucleic acids during such amplification procedures.

In previous art, ethidum bromide has also been modified through thecenter ring by attaching other intercalators (U.S. Pat. No. 5,646,264)and fragments of intercalators (U.S. Pat. Nos. 5,582,984 and 5,599,932)for improved performance of binding to double-stranded DNA. Modificationgroups similar to those disclosed in U.S. Pat. No. 5,582,984 have alsobeen added to the central ring of ethidium bromide to improveperformance with RNA (U.S. Pat. No. 5,730,849). In light of the resultswith the meta-EthD, it is disclosed that the modifications in U.S. Pat.Nos. 5,646,264; 5,582,984; 5,599,932; and 5,730,849; all of which areincorporated by reference, may also be used to synthesize novelcompounds by replacing the center ring with the phenyl ring as anattachment site. These may also be used in many of the applicationspreviously described for ethidum bromide, ethidium bromide dimers,ethidum bromide heterodimers, modified ethidium bromide compositions andother intercalators.

9. Novel Chemiluminescent Reagents

In another embodiment of the present invention, novel 1,2-dioxetanescompounds are disclosed that when used as substrates for selectedenzymes result in an intramolecular reaction between two groups attachedto different sites of an aromatic ring thereby leading tochemiluminescent signal generation. In another aspect of the presentinvention, the novel 1,2-dioxetanes compounds are disclosed that aresubstrates for modification enzymes rather than degradative enzymeswhere the modification event can lead to chemiluminescent signalgeneration.

a. Enzyme Dependent Interactions Between Two Groups Attached toDifferent Sites on a Cyclic Ring

In another aspect of the present invention, novel 1,2-dioxetane reagentsare disclosed that comprise two groups attached to different sites of acyclic ring where after catalysis by an appropriate enzyme, the reagentundergoes an intramolecular reaction thereby leading to chemiluminescentsignal generation. The reagents of this aspect of the present inventionhave the structure:

where Q comprises a cycloalkyl or polycycloalkyl group located on oneside of the dioxetane and R1 and R2 are located on different sites of acyclic ring that is bonded to the other side of the 1,2-dioxetane. Z cancomprise hydrogen, alkyl, aryl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl, or cycloheteroalkyl groups. In a preferred embodiment, Qcomprises an adamantyl group. In another preferred embodiment, the twosites where R1 and R2 are attached are adjacent to each other on anaromatic ring. R1 comprises a chemical group that is a substrate for anenzymatic activity. In the presence of the appropriate enzyme, R1 iscatalytically converted into R1* which comprises a chemically reactivegroup G1. R2 is attached to the ring through an oxygen atom andcomprises a chemical group G2 that is capable of interacting with the G1group that is produced by the conversion of R1 into R1*. Due to therigidity imparted by the ring, G1 is in close proximity to G2 therebyendowing the interaction to take place with favorable kinetics. Thisinteraction leads to formation of an unstable dioxetane therebyproducing chemiluminescence.

R2 can comprise an aliphatic group, substituted aliphatic group, anaromatic group or any combination of the foregoing. In the cases whereR2 comprises a substituted aliphatic group, the substituents can behalogens, nitrate, sulfur or nitrite. The aliphatic group can besubstituted at one position or in several positions. The substituents ateach position can be the same or different.

As described above, after the enzymatic conversion of R1 into R1*, achemically reactive group G1 is formed. Chemically reactive atoms thatmay find use as part of G1 may include but not be limited to nitrogen,sulfur or oxygen. Enzymes that may find use with present invention caninclude but not be limited to amidases, esterases,acetylcholinesterases, acid and alkaline phosphatases, decarboxylases,lipases, glucosidase, xylosidase, fucosidase, trypsin and chymotrypsin.Enzymatic substrates that may find use as constituents of R1 can includebut not be limited to amides, esters, phosphates, carboxylic acid, fattyacids, glucose, xylose, fucose or amino acids.

Although it is not essential, R1 can also be designed such that afterthe enzymatic conversion of R1 into R1* the interaction between G1 andG2 creates a 5 or 6 membered ring which is known to be an especiallystable conformation. An example of formation of such an intermediate isshown below using oxygen as the connection of R2 to an aromatic ring:

As shown above, the intermediate structure can undergo an internalsubstitution reaction that transfers the G2 group to the G1 group of theR1* moiety thereby releasing the oxygen and creating an unstable phenoxyion leading to an unstable form of dioxetane and production of achemiluminescent signal. The juxtaposition of the G1 and G2 groupscaused by locating each group on a segment of a rigid structure shouldallow efficient interaction and subsequent substitutions andrearrangements to form the light producing intermediate after productionof G1 by enzymatic activity.

b. Chemiluminescence Generation Derived from Modification Enzymes

In another aspect of the present invention novel 1,2-dioxetanederivatives are disclosed in which the triggering event that leads tothe decomposition and production of chemiluminescent signal is an enzymemodification of a specific group of the structure. This is in directcontrast to previous examples in which the triggering event is thecleavage of a substituent. In a preferred mode, the modification of thesubstitutent is dependent upon an enzymatic reaction. An example of sucha composition is given below:

In the diagram above, Q and Z are defined as described previously and Rcan comprise a chain of atoms consisting of C, N, O, S or any otheratoms required. R can also comprise saturated or unsaturated groups.Furthermore, R′ can include but is not limited to alcohols or carboxylicgroups. The modification reaction that can lead to light productionreaction can include but not be limited to oxidation and reductions.Enzymes that can be used in this aspect of the present invention caninclude but not be limited to oxidases and reductases.

A representative example of this process is given below where adioxetane derivative comprising a terminal alcohol is enzymaticallyconverted to an aldehyde by the action of alcohol dehydrogenase.

This resulting product can then undergo β elimination that then resultsin the unstable phenoxide ion that triggers the decomposition of the1,2-dioxetane resulting in the chemiluminescent signal.

10. Real-Time Signal Generation

The present invention discloses a method of signal generation that canbe used for labeling either discrete nucleic acids or a library ofmultiple sequences. The present invention provides methods andcompositions for specifically labeling analytes of interest in thepresence of other nucleic acid sequences. The present invention may alsobe used for the detection of the presence and/or amount of nucleic acidsof interest during the course of using such nucleic acids as templatesfor further nucleic acid synthesis. This can be carried out either bypost-synthesis analysis or real-time analysis during the course of suchsynthesis. In the present invention, nucleic acids are synthesized thatcomprise at least one first element of an energy transfer pair and atleast one second element of an energy transfer pair. When a first energytransfer element is capable of acting as an energy donor, the secondenergy transfer element is capable of acting as an energy transferacceptor. Conversely, the first element can be an energy transferacceptor and the second element can be an energy donor. This secondelement comes into association with the first element by virtue ofeither being incorporated into the same nucleic acid strand thatcomprises a first element or by binding to a nucleic acid strand. In theabsence of nucleic acid synthesis or a binding event, there is little orno energy transfer from the donor to the acceptor. However, by theappropriate designs, the present invention allows energy transfer from adonor to an acceptor during or after nucleic acid synthesis.

Various embodiments of the present invention use labeled primers,probes, nucleotides, nucleic acid binding agents and solid supports assources of energy transfer elements. In the present invention, a probeand a primer share the common characteristic of binding to complementarysequences with the proviso that a primer has the additional property ofbeing able to be extended. Nucleic acid constructs may also be used inthe present invention as primers, probes or templates. In the presentinvention a nucleic acid construct comprises a nucleic acid withsequences that are either identical or complementary to all or a portionof a nucleic acid of interest and may further comprise at least onenon-natural or artificial element.

Examples of non-natural or artificial elements that could comprise anucleic acid construct can include but not be limited to promotersequences, capture sequences, identity tag sequences, consensussequences, protein binding sequences, artificial primer bindingsequences, modified nucleotides, nucleotide analogues, abasic sites,labels, ligands, peptides and proteins. Furthermore nucleic acidconstructs may comprise analytes. These analytes can be individualspecific sequences or a library of sequences. They may be the originalanalyte itself or a copy thereof. They can be derived from chromosomes,episomes or fragments thereof. Examples of episomes can include but notbe limited to plasmids, mitochondrial DNA, chloroplast DNA and viruses.

a. Energy Transfer between Labeled Primers

In one embodiment of the present invention, the first and second energytransfer elements are components of at least two primers or nucleic acidconstructs that can be extended in the presence of appropriate nucleicacids. At least one of these primers or nucleic acid constructs willcomprise sequences that are complementary to sequences that are presentin a portion of a nucleic acid of interest. At least one other primer ornucleic acid construct will comprise sequences that are identical tosequences that are present in another portion of the nucleic acid ofinterest. In this way, a nucleic acid of interest can be used as atemplate for binding and extension of the primer or nucleic acidconstructs. Separation or displacement of the extended primer from thetarget allows the target strand to be used for another primerbinding/extension event. In addition the extended primer can itself beused for a primer binding/extension event. Thus one would create aproduct that comprises two extended primers hybridized to each other. Inthis aspect of the present invention, the primers used for the precedingsequential primer binding/extension events comprise either a firstenergy transfer element or a second energy transfer element. If theprimers in each strand are in sufficient proximity to each other, theywould be capable of allowing an energy transfer event from a donor to anacceptor. This process can be used to create a double-stranded labelednucleic acid. Of especial utility for diagnostic purposes, the extent ofthe signal generated by this process can be used to identify thepresence and quantity of the particular nucleic acids used as templates.

The amount of signal can also be increased by introduction ofamplification processes. For instance, the use of a primer for eachstrand of a desirable nucleic acid target is the basis of many targetamplification procedures where strand extension of each primer generatestemplates for further synthetic events. These methods can depend upondiscrete steps such as those employed in PCR (U.S. Pat. No. 4,683,202)or they can be continuous isothermal methods such as SDA (U.S. Pat. Nos.5,270,184 and 5,455,166) and Loop Mediated Amplification (U.S. patentapplication Ser. No. 09/104,067; and European Patent Application No. EP0 971 039 A) (all the foregoing incorporated by reference). Thus,although the present invention can be used for post-synthesis assessmentof the amount of synthesis of appropriate nucleic acids, it can also beused during the multiple synthetic steps that take place during thecourse of amplification, i.e real time analysis. Amplification can becarried out under the same conditions used in the absence of labeledprimers or an additional step can be included that can increase theefficiency or selectivity of signal generation. For instance, for realtime analysis of an isothermal reaction, monitoring can take placecontinuously or at chosen intervals. In the latter method, an extra stepcan be carried out where either a sample is removed for analysis or athermal step is introduced that promotes signal generation or reading ata particular state but does not substantially interfere with thecontinuation of the reaction.

In previous art, the design of primer locations for double strandedsynthetic nucleic acid products for diagnostic purposes has been to havethe primers for each strand located sufficiently apart that additionalsequences are incorporated in between them that can be used forhybridization with probes or characterization by restriction enzymes.Thus, the sequences of double stranded synthetic nucleic acid productswould be derived from two sources. First, there would be intrinsicsequences derived from the primers and their complements. These will bepresent independent of what particular target segment was used as atemplate. Secondly, there would be the sequences between the primersegments. These would be totally dependent upon the nature of theparticular nucleic acid segment used as a template for nucleic acidsynthesis. Depending upon the nature of the design of the primers, theconditions of the reaction and the particular nucleic acid sequences inthe sample used in the amplification reaction, only a particulardesirable sequence may be synthesized or other non-desirable sequencesmay also be synthesized. For diagnostic purposes, the segments betweenthe primers have then been used as a target for a labeled probe togenerate a signal that would be dependent upon the presence and amountof only the desirable sequences.

In this particular embodiment of the present invention, the requirementfor extended sequences between the primer segments is abrogated sinceprobes are not used for the detection of the amplification product. Infact, the present invention discloses that a proximity between theprimers at each end of an amplicon is a desirable arrangement that canbe used for a novel means of signal generation. By including a firstelement into a primer for one target strand and a second element into aprimer for the complementary target strand, proximity of these twoprimers in a double stranded amplicon allows energy transfer to takeplace from the element that acts as a donor to the element that acts asan acceptor even though each element is on a different strand.

As described previously, various amplification systems that are basedupon a series of primer extension reactions that result in doublestranded amplicons with incorporated primers will be able to enjoy thisparticular embodiment of the present invention. For instance, FIG. 3shows potential amplification products for a) PCR and b) SDA. Details ofthe processes that can be used for these amplification methods can beseen in numerous publications including the original patent for each ofthese methods (Mullis et al. in U.S. Pat. No. 4,683,202 and Walker etal., in U.S. Pat. Nos. 5,270,184 and 5,455,166; incorporated herein byreference). Even though these methods employ different principles, thepresence of a labeled primer or nucleic acid construct in each strand ofan a double-stranded nucleic acid allows the use of the presentinvention. In addition, other methods that have been previouslydisclosed may find use with the present invention including multiprimeramplification (U.S. patent application Ser. Nos. 08/182,621; filed Jan.13, 1994; 09/302,816, filed Mar. 31, 1998; and 09/302,818, filed Feb. 3,1998; and 09/302,817, filed Apr. 16, 1999) and amplification withinverted oligonucleotides (U.S. Pat. No. 5,462,854) all of which arehereby incorporated by reference.

As described previously, this particular embodiment of the presentinvention depends upon a proximity between the primers or nucleic acidconstructs on each strand. In terms of an extended strand made from afirst primer or nucleic acid construct, this can also be described asthe proximity between the segment derived from the incorporated firstprimer or nucleic acid construct and the segment that can be used as abinding site for the second primer or nucleic acid construct tosynthesize the complementary strand. For instance, proximity can beachieved by having these two segments being immediately adjacent to eachother on an extended strand. In such a case, the nucleic acid sequencesof the extended strands would be entirely derived from the sequences ofthe primers or nucleic acid construct and their complements. To depictthis more clearly, an arbitrary sequence is shown in FIG. 4 withpotential primer arrangements that could be used in the presentinvention. In FIG. 4(A) the sequences chosen for primers are immediatelyadjacent to each other on each strand. Alternatively, there can be a gapbetween the primer segment and the primer binding segment on one strandas long as there is sufficient proximity for energy transfer between thedonors and acceptors in the amplification product. An example of alonger spacing using the same target sequence is shown in FIG. 4(B).

It should also be noted that in addition to allowing a novel system ofsignal generation, the reduction of the amplicon size such that itcomprises little more than primer binding regions should conferadvantages over the more traditional longer amplicons. These shouldinclude shorter extension times, sharper melting points, and overallhigher efficiency in each round of amplification since the amount ofsynthesis is of a minimal nature. Also, the choice of appropriate energytransfer elements and detection systems can allow multiplexamplification to monitor more than one target sequence.

In the presence of the appropriate target sequences, signal generationshould increase as more labeled primers become incorporated intodouble-stranded nucleic acids. This signal generation should be specificand proportional to the presence of appropriate target molecules in thesample. Thus, in the absence of nucleic acid synthesis, there should belittle or no energy transfer between donor and acceptor molecules sinceeach element is located on a separate primer or nucleic acid construct.Secondly, signal generation can be carried out under reaction conditionsthat allow little or no nucleic acid synthesis in the absence ofappropriate target templates. One way that this can be carried out is byappropriate design of the primers themselves such that primer-dimerformation is minimized, for instance by selecting primer sequences thathave no overlap between their 3′ ends. On the other hand, if non-targetnucleic acids are present that have sequences present which have somesimilarity to the primer binding sequences, nucleic acid synthesis maytake place, but the nucleic acid product is unlikely to have the primersincorporated into the appropriate lengths for energy transfer to takeplace. Another way that target-specific signal generation can beincreased is by the use of what has been termed “nested PCR”. In thismethod, the majority of amplification is carried out by a second set ofprimers that flank the labeled primers. This is shown in FIG. 3(C). Thelabeled primers can be present in reduced amounts, require differentannealing conditions or be used in separate short amplificationreactions. This should reduce the involvement of the labeled primers inamplification of either primer-dimers or non-target sequences. In thisparticular instance, it may even be possible to successfully generatetarget dependent signals with labeled primers or nucleic acid constructsthat have some degree of overlap between their 3′ ends. Lastly, targetindependent products should have a different length and/or basecomposition thereby allowing a differentiation between target specificdouble-stranded nucleic acids and inappropriate products by theirthermal profiles. As described previously, this profile can be obtainedas part of the process or a separate step may be introduced to obtainsuch a profile.

Although this particular embodiment of the present invention has beendescribed in terms of incorporation of nucleotides, there are also meansfor extending primers that depend upon the addition of polynucleotidesrather than individual nucleotides. As such, two of these methods, LCR(U.S. Pat. No. 5,494,810) and GAP-LCR (U.S. Pat. No. 6,004,286), mayalso enjoy the benefits of the present invention. These methods dependupon the use of two sets of adjacent oligonucleotides where each set iscomplementary to one particular strand of a target nucleic acid. In thepresent invention, a first energy transfer element will be in one ormore oligonucleotides complementary to one strand and a second energytransfer will be in one or more oligonucleotides complementary to theother strand. An illustration of the use of the present invention withthis method is shown in FIG. 3(D).

b. Energy Transfer between a Labeled Primer and Nucleotide(s)

In another embodiment of the present invention, one or more primers ornucleic acid constructs that comprise a first energy transfer elementare used in conjunction with at least one nucleotide that comprises asecond energy transfer element. After target template directed additionof nucleotides to the primer or nucleic acid construct, energy transfercan then take place by interaction between a first energy transferelement in one primer or nucleic acid construct and a second energytransfer elements in an incorporated nucleotide. The labeled primer ornucleic acid construct and the labeled nucleotide or nucleotides can beon the same strand if only a single primer or nucleic acid construct isused during primer extension events. Linear amplification can also becarried out where the primer or nucleic acid construct is used forsuccessive rounds of binding/extension events.

On the other hand as described previously, the inclusion of one or moreprimers or nucleic acid constructs that can use the extended primers orextended nucleic acid constructs as templates can allow furthersynthesis. In this case, the second energy transfer elements that areintroduced by nucleotide incorporation can be in both the extendedstrand and its complementary copy. FIG. 5 shows potential amplificationproducts made by various amplification processes that illustrate thisparticular embodiment of the present invention. In FIGS. 5(A), 5(B) and5(C), one or more of the primers used for amplification contain anenergy transfer element. Although this Figure shows the acceptor element(A) being present in primers and the donor elements (D) being present inthe nucleotides incorporated during amplification, the oppositearrangement may also be used. In this particular aspect of the presentinvention, the spacing between the primers can be of any desired lengththat is appropriate for carrying out the amplification.

As described previously, various methods may be employed to selectivelygenerate signal from only appropriate targets. These can include primerdesign, thermal profiling of double-stranded nucleic acids and nestedamplification. This particular embodiment of the present invention isalso amenable to multiplex formats. For instance, if various primers areused such that more than one extended primer species is synthesized,they can be distinguished from each other by using a common energytransfer donor in the nucleotides and different energy transferacceptors in each of the primers. Each of the individual nucleic acidproducts can then be identified by the spectral characteristics of theacceptor on the primer.

Previous art has described the dual use of both a primer that comprisesa first energy transfer element and a dideoxyribonucleotide thatcomprises a second energy transfer element (Kwok and Chen, U.S. Pat. No.5,945,283). The present invention differs from this art in usingnucleotides that are not strand terminators in the reaction mix therebya) allowing for the possibility of multiple incorporation events and b)allowing sufficient synthesis that the extended strand could be used asa template for synthesis of a complementary nucleic acid if desired.

c. Energy Transfer between Labeled Nucleotides

In another embodiment of the present invention, it is disclosed thatsignal generation can take place during synthesis with labelednucleotides only. In this particular embodiment, synthesis is carriedout in the presence of at least one nucleotide that comprises a firstenergy transfer element and at least one nucleotide that comprises asecond energy transfer element. The nucleotides that comprise first andsecond energy transfer elements may be the same nucleotide, for instanceby using a mixture of dUTP, where some are labeled with an energytransfer donor and some are labeled with an energy transfer acceptor. Onthe other hand, they may be different nucleotides, for instance by usinga mixture that has dUTP labeled with an energy transfer donor and dCTPlabeled with an energy transfer acceptor.

As described above, incorporation of nucleotides that comprise first andsecond energy transfer elements can take place during a single round ofstrand extension, multiple rounds of extension of one strand for linearamplification, or by the provision of at least one second primer ornucleic acid construct for exponential amplification. FIG. 5(D) shows aPCR amplification product where both donors and acceptors have beenincorporated through labeled nucleotides. In the absence ofincorporation, there will be little or no energy transfer between onenucleotide to another. However, once they have been incorporated intonucleic acid strands, they are in position to be able to allow energytransfer to take place. This can be through intrastrand interactions inthe same strand or through interstrand interactons between nucleotideson complementary strands. A particular nucleotide base may consistentirely of labeled nucleotide or there may be a mixture of labeled andunlabeled nucleotides.

Although methods such as PCR and SDA produce double-stranded ampliconsas their major product, there are systems such as NASBA that alternatebetween double-stranded DNA and single-stranded RNA forms. In theseamplification methods, the present invention finds use by providingeither energy transfer labeled deoxyribonucleotides for labeling the DNAor energy transfer labeled ribonucleotides for labeling RNA products. Inthe latter case the presence of both donor-labeled and acceptor-labeledribonucleotides in the RNA strands would allow intrastrand energytransfer. As described previously, various methods may be employed toselectively generate signal only from appropriate amplicons. These caninclude primer design, thermal profiling of double-stranded ampliconsand nested amplification. Additionally, since signal generation in thisparticular embodiment of the present invention is derived from theenergy transfer between incorporated nucleotides, the method describedby Singer and Haugland (U.S. Pat. No. 6,323,337 B1) can also be usedwhere the primers comprise energy quenchers. Quenchers that may be usedfor this purpose can include non-fluorescent derivatives of fluorescein,rhodamine, rhodol or triarlylmethane dyes as described by Singer andHaugland (op. cit.).

d. Energy Transfer between a Fluorescent Intercalator and a LabeledPrimer or Nucleotide(s)

The previous embodiments of the present invention have utilized primersand nucleotides as energy transfer elements. Another embodiment of thepresent invention discloses that nucleic acid binding agents can be usedas energy transfer elements after strand extension. It has previouslybeen described in U.S. Pat. No. 4,868,103 that energy transfer can beused in a hybridization assay that involves a labeled probe and anintercalator. In contrast to this art, a labeled primer or nucleic acidconstruct with a first energy transfer element is extended to synthesizenucleic acids that can be bound by a nucleic acid binding agent thatcomprises a second energy transfer element and is substantially sequenceindependent. Binding can take place while the extended strand is stillbase-paired with its template or after separation from the template i.e.the extended strand is in double-stranded or single-stranded form. Thenucleic acid binding agent can be a protein or a chemical that has ahigh affinity for nucleic acids. An example of proteins that may finduse with the present invention may include but not be limited to T4 gene32 protein, SSB protein, histones and antibodies. The T4 gene 32 proteinand SSB protein have affinities for single-stranded nucleic acids andthe histones have an affinity for double-stranded nucleic acids.Antibodies specific for nucleic acids and for RNA/DNA hybrids have beendescribed in the literature (U.S. Pat. Nos. 6,221,581 and 6,228,578).Methods for attaching fluorescent labels to proteins have been widelydescribed in the art. An example of a chemical that has a preferentialaffinity for single strand nucleic acids can include but not be limitedto SYBR™ Green II. An example of a chemical that has a preferentialaffinity for double-stranded nucleic acids can include but not belimited to intercalators. Examples of intercalators that may find usewith the present invention can include but not be limited to Acridine,Ethidium Bromide, Ethidium Bromide Homodimers, SYBR™ Green I, TOTO™,YOYO™, BOBO™ and POPO™. The binding agent can comprise a energy transferelement directly or indirectly. Proteins labeled with an energy transferelement would be examples of indirect means. The intercalators listedabove would be examples of direct means.

Also, energy transfer to or from nucleic acid binding agents can becarried out by labeled nucleotides instead of labeled primers ifdesired. When the nucleic acid is in double-stranded form, thisembodiment of the present invention can take advantage of the ability ofsome intercalators to have enhanced fluorescence upon binding todouble-stranded nucleic acids. As has been mentioned earlier, thiseffect has been used by itself to monitor real time nucleic acidsynthesis during amplification reactions. However, when used alone, thismethod suffers from the amount of background exhibited by the dye aloneor by dye binding to single-stranded primers. This deficiency may beovercome by the present invention since unbound dye should be unable toundergo an energy transfer interaction with unincorporated labelednucleotides. As such, the present invention should enhance theselectivity of signal generation compared to using a labeled nucleicacid binding agent alone.

As described previously, various methods may be employed to selectivelygenerate signal from only appropriate target molecules. These caninclude primer design, thermal profiling of double-stranded ampliconsand nested amplification.

e. Energy Transfer between a Labeled Probe and Nucleotide(s)

There may be circumstance where the specificity contributed by nucleicacid probes may desirable. Therefore, another aspect of the presentinvention, discloses novel means of signal generation where at least onenucleic acid probe that comprises a first energy transfer element isused in conjunction with either nucleotides that comprise second energytransfer elements. Previous art has described the use of an energytransfer labeled primer and an energy transfer labeled sequence specificprobe (Wittwer et al. in U.S. Pat. No. 6,174,670). In contrast to thisart, the present method is not constrained to the use of a probe that isin proximity to the primer alone but allows the use a probe designed toanneal to any location on the nucleic acid strand that is desirable. Inaddition, the present invention conveys the ability to use multipleenergy transfer probes by using various segments of the extended nucleicacids as probe targets. Thus, when using nucleotides that compriseenergy transfer elements, signal generation should take place afterhybridization of labeled probes to the labeled nucleic acid strand.

For instance, after strand-extension, the separation or removal of thetemplate strand can allow the binding of a probe to a single-strandedextended strand and thereby allowing energy transfer to take placebetween first energy transfer elements in the probe and second energytransfer elements that have been incorporated into the extended strand.Energy transfer to or from first elements in the probe can be derivedfrom the segments that are hybridized to the probe, or if they are insufficient proximity, they may be from adjacent single-stranded regions.Another illustrative example of the present invention makes use ofloop-mediated amplification (U.S. patent application Ser. No.09/104,067; European Patent Application Publication No. EP 0 971 039 A).One of the structures generated by this system is a single-stranded loopadjacent to a double stranded stem. Therefore, as disclosed in thepresent invention, a probe can be bound to sequences in the loop regionand undergo an energy transfer reaction with incorporated nucleotides.

In this embodiment of the present invention, specificity is generated bytwo factors. First, the strand extension events should be dependent uponthe specificity of the primer binding/extension reactions themselves.Secondly, the probes are blocked at their 3′ ends and only participateby binding to appropriate sequences when these are synthesized as aresult of the primer extension reactions.

The various embodiments of the present invention that have beendisclosed above can be used in homogeneous assay systems. However, thereare also advantages that are offered by the use of solid supports withthe present invention. Fixation to solid supports can take place priorto initiation of extension reactions, during strand extensions, andafter completion of strand extensions. Examples of solid supports thatmay find use with present invention can include but not be limited tobeads, tubes, microtiter plates, glass slides, plastic slides, microchiparrays, wells and depressions. Fixation to the support can be carriedout either directly or indirectly. As an example of indirect fixation, acapture agent may be attached to the solid support. This capture agentmay be a nucleic acid with sequences that are complementary to sequencesthat are present in a primer, a nucleic acid construct, an analyte or acopy of an analyte thereby allowing fixation through hybridization.Another example of a capture agent could be an antibody that has anaffinity for a nucleic acid. (U.S. Pat. Nos. 4,994,373; 4,894,325;5,288,609; 6,221,581 B1; and 6,228,578; all of which are incorporated byreference).

As an example of direct fixation, many of the previous embodimentsemploy a primer for strand extension. Therefore if desired, theseprimers could be covalently attached to a solid support prior tocarrying out any extension reactions. In the presence of the appropriatenucleic acids, strand extension can then occur as described previouslythereby resulting in strand extension products that are also directlyattached to the solid support. First and second energy transfer elementscan be in the primers fixed to the solid support and/or they can be innucleotides, probes or nucleic acid binding agents as described in thevarious embodiments disclosed above. An illustrative example of thiswould be the use of amplification on a microarray as described inRabbani et al., U.S. patent application Ser. No. 09/896,897, filed onJun. 30, 2001 (incorporated by reference) with at least one set ofprimers at a locus comprising a first energy transfer element andnucleotides comprising second energy transfer elements. With theappropriate apparatus, each locus on the chip could then be separatelymonitored for the extent of synthesis during the course of theamplification. Another example of direct attachment would be the use ofprimers that comprise ligands and solid supports that comprise theappropriate ligand receptors. An illustrative example of thisarrangement could be the use of poly T primers that have been labeledwith biotin and using beads that are coated with avidin or strepavidinfor fixation. Nucleotides that comprise first energy transfer elementscan be incorporated into a cDNA copy made with the poly T primer and thecomplex of a cDNA bound to its RNA template can bind intercalators thatcomprise second energy transfer elements. In this example, attachment ofthe primers to the beads can take place before, during or after thestrand extensions.

f. Solid Supports Comprising Energy Transfer Elements

In addition, a solid support is not relegated to only a passive role. Inanother embodiment of the present invention, a solid support comprises afirst energy transfer element. Fixation of a nucleic acid to the supportcan then bring a second energy transfer element into sufficientproximity for a signal to be generated. In this embodiment of thepresent invention, the second energy transfer element can be part of anucleotide, primer, probe or nucleic acid binding agent. As anillustrative example, a matrix is made with a selection of nucleic acidprobes on an array. The matrix is then treated such that first energytransfer elements are fixed to the surface as well. In the presence ofappropriate templates or analytes, synthesis of a group of nucleic acidswith second energy transfer elements is then carried out as describedpreviously. Hybridization of the labeled nucleic acids to the arraybrings the second energy elements into proximity with the surface boundfirst elements. Signal should then be generated by energy transfer thatcorresponds to the amount of nucleic acids that are bound to aparticular locus on the array. An illustration of this principle as wellas some of the other embodiments described previously is depicted inFIG. 6. Solid supports with capture elements can also be used in methodswhere no extension reactions are required. For instance, a solidsupport, a capture oligo or an antibody specific for nucleic acids cancomprise a first element. Signal generation can then occur after bindingof an unlabeled analyte in either single stranded or double strandedform to the support where the presence of the second element is dictatedby the presence and amount of the analyte that becomes bound to thesupport. For instance, the second element can be part of the complex inthe form of a probe or a nucleic acid binding agent.

g. Previous Processes (a through f) Used for Labeling

Generation of signal from first and second energy transfer elementsafter target dependent strand synthesis can be used for the purpose ofdetecting the presence or amount of a nucleic acid of interest in asample. When signal generation is dependent upon the specificity of thepriming events, the procedures are carried out under conditions wheretarget independent primer extension is minimized or as describedpreviously, methods are included that can distinguish between targetderived nucleic acids and spurious strand extension products. On theother hand, inclusion of a step that uses the discretionary power ofnucleic acid hybridization can limit or even obviate the need for primerspecificity. For example, an entire library of poly A mRNA sequences canbe converted into a library of cDNA copies by the use of a universalpoly T primer. The library of cDNA strands can then be indiscriminatelyused as templates for second strand synthesis. Inclusion of a promoterinto either the first strand primer (U.S. Pat. No. 5,891,636) or secondstrand primer (Rabbani et al., U.S. patent application Ser. No.09/896,897, filed on Jun. 30, 2001) (both documents incorporated byreference) allows synthesis of multiple RNA copies of each individualoriginal mRNA. When either this product or a cDNA copy is labeled,hybridization with a microarray of nucleic acids can be used todetermine the amounts of any particular species in the original sample.The present invention can be employed with such methods by includingfirst and second energy transfer elements in primers, nucleotides,probes, nucleic acid binding agents or the matrix itself.

On the other hand, the nucleic acids of interest may be supplied by theuser in known quantity and the embodiments of the various inventiondisclosed above may be used to synthesize labeled probes. For instance,a single purified species of a nucleic acid of interest might beprovided as a template for labeling reactions with one or two primers.When a discrete group of varied nucleic acids is desired to be labeled,the primer sets can be expanded accordingly. Labeled probes can then becreated by inclusion of first and second energy transfer elements by anyof the means disclosed previously. These probes can then be used toidentify the presence or quantity of unlabeled nucleic acids in samplesin any of a variety of formats that are well known to those skilled inthe art.

h. Analytes as Primer Extension Substrates

It should also be noted that the nucleic acids of interest or copies ofthe nucleic acids of interest may also be used directly in strandextension reactions either as substrates for terminal transferaseaddition, ligation or by acting as primers. For example, terminaltransferase can be used for the template independent addition of firstenergy transfer elements onto the 3′ ends of either individual nucleicacids or a library of nucleic acids. Second energy transfer elements canthen be included as part of the terminal addition reaction or they cancomprise primers, probes, nucleic acid binding agents or solid supports.In another illustrative example, restriction digestion of DNA can befollowed by terminal addition of a mixture of nucleotides with first andsecond energy transfer elements. Various individual species of nucleicacids can then be hybridized to various capture sequences on discreteloci of an array to measure the presence or amount of individual labeledsequences.

It is also a subject of the present invention that analytes or copies ofanalytes can be used as primers in template dependent labelingreactions. In this context, incorporation in itself may be used as anassay since template directed synthesis should be dependent upon thepresence of discrete sequences in the analytes that correspond to theircomplements in appropriate hybridized templates. Thus a desired nucleicacid sequence can be specifically labeled in the presence of othernucleic acids that may be present in a mixture. Although at least onesegment of the template is designed to match the desired analytesequence, the segment of the template that is used direct the sequencesadded onto the analyte can be either a natural sequence or an arbitrarysequence.

As an illustrative example of this method, a library of polyA mRNA canbe labeled in the presence of total RNA by using a probe that comprisesa first segment that comprises Poly T and a second segment that can beused as a template. When bound to the poly A sequences of the mRNAthrough the first segment, the 3′ end of the mRNA can be extended usingthe second segment as a template. The nucleotides that are incorporatedusing the second segment as a template can be either labeled orunlabeled. Examples of artificial sequences that may find use as secondsegments can include primer binding sites, RNA promoter sequences.Another illustrative example is a series of probes that comprise firstsegments complementary to the 3′ ends of discrete bacterial RNA species.For each particular species-specific segment, a discrete templatesequence can be used. After specific priming by the RNA present in asample, evaluation can be carried out with an array that has capturesequences complementary to the extended sequences thereby separating outthe individual extended RNAs. And lastly, cDNA copies can be made from apool of mRNA using standard techniques. Each cDNA that represents a fullcopy of the original mRNA should have a discrete 3′ end that representsthe 5′ end of the original mRNA. Template/Probes could then be used foreach cDNA that is desired to be quantified. In these illustrativeexamples of using an analyte as a primer in template dependentextensions, labeling may take place by incorporation of a singlelabeling species as described in previous art or the methods that havebeen disclosed above may be employed using first and second energytransfer elements.

11. Fragmentation and/or Incorporation of Desirable Nucleic AcidSegments

a. Template Dependent Addition of Desirable Nucleic Acid Sequences tothe Ends of Analytes

It is another aspect of the invention to provide novel compositions andmethods for the template dependent addition of desirable nucleic acidsequences to analyte or target nucleic acids. In prior art, poly Asequences have been relied upon as the basis for most methods ofmanipulation of mRNA. Furthermore, the utility of mRNA has derived fromits use as a template to carry out any and all such manipulations. Forinstance, Poly A has been used as a primer binding site for making cDNAcopies and carrying out linear or exponential amplification of mRNA.However, as described previously, this feature is not universally sharedamong all RNA targets. Furthermore, it is a selective feature for the 3′end of mRNA. In contrast to this art, the present invention overcomesthe limited scope of analysis of nucleic acids by viewing and using ananalyte or target nucleic acid not as a template but as a substrate forstrand extension, i.e. as a primer. As such, the nucleic acid constructsthat are provided for these processes are not used as primers but ratherthey serve as templates to enable analyte or target nucleic acids toincorporate any arbitrary sequence that is desired by the user. Suchsequences can comprise promoters, primer binding sites or signalgenerating moieties.

In the present invention, methods that may be directed for use withnucleic acid analytes may also be used with any desirable nucleic acidtarget as well. Analytes can comprise single desirable sequences or theymay be a library of various sequences. Analyte or target nucleic acidsmay be comprised of RNA or DNA as well as copies of RNA or DNA. Theanalyte or targets nucleic acids may be extracted from biologicalsamples or they may have been produced in vitro. They may also haveundergone procedures and processes such as digestion, fragmentation,amplification, extraction and separation.

The present invention discloses that the ends of nucleic acids can behybridized to complementary chimeric nucleic acid constructs (CNACs)that comprise two segments. The first segment comprises nucleotides ornucleotide analogues that are capable of binding or hybridizing to the3′ ends of the analytes. The second segment comprises nucleotides ornucleotide analogues that can be used as a template for extension of the3′ end of the analyte. In contrast to prior art, methods are disclosedthat do not rely upon the presence of a selected sequence such as a polyA segment at the ends of the anlyte, but rather the present inventiondiscloses methods where any and all sequences that may be present at the3′ end of an analyte or library of analyte are sites for binding andtemplate-dependent extension reactions. In the present invention,template-dependent strand extension can take place either byincorporation of individual nucleotides (polymerization) or by additionof pre-synthesized oligonucleotides (ligation). It should be pointed outthat although the inventions are commonly described in terms of 3′extension since this is a characteristic of polymerase driven processes,when ligation is used instead, the 5′ end is also a suitable substratefor template dependent strand extension. The ability to introducearbitary nucleic acid sequences into the 3′ end of a target or analytenucleic acid provides a simple and powerful vehicle for transforming ananalyte into a probe or a nucleic acid construct that could be used forfurther manuipulations. A library of nucleic acids with varioussequences can also be converted into a library with universal sequencesthat could be later used for further manipulation directly in acontrolled and measured manner for signaling purposes, priming events,capture events or amplification events.

In one particular embodiment of the present invention, a set of CNACs isused where the first segment comprises all the potential permutations ofnucleotide sequences. Thus, if the first segments of the set of CNACscomprise 6 variable nucleotides, the first nucleotide (N₁) can be G, A Tor C, the second nucleotide (N₂) can be G, A T or C etc. and the setitself will comprise 4⁶ (4,096) different CNACs. In this sense, it hassimilarity to the use of random primers for synthesis of nucleic acidcopies. However the present invention differs from random priming inthat the CNACs are not extended themselves (i.e., act as primers) butprovide complementary binding to the analytes such that the secondsegment of the CNAC can be used for template dependent extension of theanalyte. For this purpose, it is preferred that the ends of the CNACs beblocked. Thus although the present invention uses random sequences, theside reaction of random primers using each other as primers andtemplates is completely avoided. The present invention also differs inthat the binding of random primers to any particular site of an anlyteallows an extension event. In the present invention, it is only when theCNAC binds to a complementary sequence at the end of an analyte that anextension event takes place. Although, there will be random binding anddisassociation of the CNACs at multiple sites on the analyte strands,this is not a true equilibrium situation since there is actually adynamic favoring of binding to the ends. For instance, juxtaposition ofa 3′OH in the analyte and a complementary CNAC can bind a polymerase andform a complex that would be more stable than a CNAC bound to aninternal site. In addition to providing a longer half-life of binding ofthe CNAC to the terminus by complex formation, the complex generates aneven more stable form by extending the analyte, thereby increasing thenumber of bases that are complimentarily base paired. Thisdisequilibrium can be carried out in an isothermal reaction, or ifpreferred, the reaction temperature can be raised to promotedissociation of CNACs from non-productive binding sites followed by areturn to the same reaction temperature to promote another round ofbinding of CNACs to analytes. If desired, these variable conditions canbe recycled multiple times to optimize the amount of analyte ends thatundergo template-dependent addition.

It is a further objective of the present invention to disclose novelcompositions and methods that utilize CNACs synthesized with universalbases i.e., bases that can base pair with more than one complementarybase. Nucleotides or nucleotide analogues that comprise universal basescan contribute stability without adding complexity. Therefore, in thisaspect of the present invention, a novel CNAC is disclosed thatcomprises two segments as described above, but instead of usingpermutations of nucleotides, universal bases that lack sequencespecificity are used in the first segment. For instance, an example wasgiven above with a set of CNACs that comprised permutations in 6positions thus requiring 4,096 different CNACs. By the use of universalbases, only a single CNAC species is required for providingtemplate-dependent addition of desirable nucleic acid sequences to anyanalytes or set of analytes irrespective of the sequences at their ends.Since universal bases do not always display a complete lack ofdiscrimination and the ability to bind to a particular nucleotide, itwould be possible and even desirable to use a set of CNACs that comprisedifferent universal bases or universal base analogs, or differentmixtures of universal bases and universal base analogs. As describedpreviously, this method can involve a self-selecting process where CNACsundergo a series of binding and dissociation events of the universalbases to random segments of the analytes until the CNAC binds to a 3′end. In this particular embodiment, base pairing at the end is not aproblem since each CNAC possesses universal base pairing capability andproductive extension should be mostly related to the relationshipbetween the 3′ end of the analyte and the second segment of the CNAC.Efficient strand extension can take place where the beginning of thesecond segment of the CNAC is aligned with the 3′ end of the analytesuch that the first base synthesized will be the complement of the firstbase of the second segment. On the other hand, universal bases also havesome capacity for use as templates and as such, hybrids where the 3′ endof the analyte is not perfectly adjacent to the junction between thefirst and second segments of the CNAC should also be able to carry outstrand extension of the analyte.

It is a further objective of the present invention to disclose novelcompositions and methods where CNACs comprise universal bases incombination with permutations of nucleotides. In this particularembodiment, the CNAC can be considered to comprise three differentsegments wherein a first segment comprises universal bases, a secondsegment represents permuted series of discrete nucleotides at one ormore positions and the third segment comprises a nucleic acid that canbe used as a template for extension of 3′ ends. Thus the presentinvention should be able to enjoy the stability without complexity ofthe universal bases in the first segment in conjunction with selectivityand further stability contributed by specific base pairing by apermutational second segment anchor. Thus, if a universal base used inthe first segment of a CNAC has approximately half of the bindingaffinity as a base pairing between normal nucleotides, a set of CNACsthat comprised 4 variable nucleotide positions and 4 universal baseswould have the same average Tm as random hexamers, but would requireonly 4⁴ or only 256 different CNACs. This is compared to the 4,096different CNACs required with a hexamer permutational first segment.Similarly a CNAC with 4 universal bases and 6 variable positions wouldcomprise 4,096 different CNACs but would have binding propertiesanalagous to CNACs with random octamer first segments that would require4⁸ permutations (i.e. 65,536 different CNACs).

Therefore, one would cover all possible permutational combinations thatcould exist in the terminal end of any analyte regardless of itsderivation, while at the same time enjoying reasonably high bindingefficiency and capabilities because the universal sequences in the firstsegment provide the additional binding stability without imposing anyfurther specificity. Thus for the same number of CNAC molecules in areaction mixture, there would effectively be a 16 times higherconcentration of CNACs that could bind to a particular 3′ end of ananalyte in the examples cited above. This should provide superiorkinetics and efficiency compared to CNACs with only permutationalsegments.

Universal bases, i.e., bases that can base pair with more than onecomplementary base, were first used in oligonucleotides to maintainstable hybridization with target nucleic acids that had ambiguity in theidentity of their nucleotide sequence. A well-known example of this isthe substitution of inosine in PCR primes (Liu and Nichols, (1994)Biotechniques 16; 24-26). Inosine has the property of being able to basepair efficiently with either G, A, T or C in a complementary strand(Kawase et al., 1986, Nucl. Acids Res. 19; 7727-7736). The meltingtemperature is less than a normal base pairing but still higher than amismatch. When used as a template, inosine is recognized as if it waseffectively G and a C is preferentially incorporated into thecomplementary copy. Other analogs of nucleotides that can act asuniversal bases have also been described. For instance,5-nitroindolenine and 3-nitopyrrole analogues have also been describedas universal bases (Loakes and Brown, 1994, Nucl. Acids Res. 22;4039-4043, Nichols et al., 1994, Nature 369; 492-493 both of which areincorporated by reference). The use of these and other universal basesare reviewed by Loakes (2001) in Nucl. Acids Res. 29; 2437-2447(incorporated by reference). The ability of universal bases to addstability without adding to the complexity of primers has been describedby Ball et al., (1998, Nucl. Acids Res. 26; 5225-5227, incorporated byreference) where the addition of 5-nitroindolenine residues at the 5′end, improved the specificity and signal intensity of octamer primersused for cycle sequencing. Thus, these and other universal bases may allfind use in the present invention.

As described above, the present invention allows any nucleic acid ornucleic acid fragment to be used for template-dependent extension andobviates dependency upon poly A tails. The desirable nucleic acid (ornucleic acid of interest) that is incorporated into an analyte strandcan transform any nucleic acid or nucleic acid fragment into a form thatprovides a primer binding sequence that can carry out functionspreviously enjoyed by polyadenylated nucleic acids. Linear amplificationcan be carried out by incorporating a promoter as the desirable nucleicacid (or nucleic acid of interest) in a CNAC and exponentialamplification can be carried out with desirable primer binding nucleicacid sequences using any of the methods previously described for poly Atargets. Additionally, it is contemplated that template-dependentincorporation of a nucleic acid into an analyte also presents theopportunity to directly label the analyte or analytes by using a labelednucleotide or oligonucleotide in the incorporation step.

Research studies have had a focus on poly A mRNAs due to itsaccessability and convenience as a substrate. The present inventionallows non-polyadenylated nucleic acids to be manipulated with the sameease of use previously accorded to poly A mRNA. Thus, the presentinvention can be used with DNA, hnRNA, snRNA, tRNA, rRNA, bacterial mRNAor any RNA lacking a poly A sequence. Even poly A mRNA may find use withthe present invention. The reliance upon the 3′ poly A tail has led to abias towards the information contained in this end. In most methods ofprior art, sequences at the other end of mRNA were still dependent uponthe efficiency with which a priming event at the 3′ took place.Accordingly, any interruptions in the copying process or a scissionbetween the 5′ end and the poly A end reduced the amount of 5′ sequencesthat were available for study or manipulation. Thus, even a single nickin a large mRNA molecules eliminated the use of the 5′ end of themolecule and numerous reports and even commercial products are dedicatedtowards the preservation of the continuity between the 5′ and 3′ ends ofmRNA during its isolation. Since the present invention discloses methodsthat are independent of poly A, fragments of poly A RNA that have becomeseparated from the poly A region remain available for use and study.

In fact, such a fragmentation process can be advantageous since allsegments of the poly mRNA can be independently and efficiently used withno bias derived from their relationship to the 3′ end. Thisfragmentation will be especially useful for hnRNA which has remained anunderutilized area of research. This neglect has stemmed from twocharacteristics of hnRNA: the lack of poly A as a handle and the verylarge average size. Although the introns that are present in hnRNa lackcoding sequences for the final gene product, there are likely to be alarge number of sequences that do not appear in the final product thatare important in control, regulation and interaction with other genesand gene products. The present invention will allow the sequencespresent in hnRNA to be as completely accessible as the polyA mRNAsequences had been previously.

Although many of the embodiments of the present invention are describedin terms of RNA analytes, it should be pointed out that many of theseprocesses can easily be applied to DNA fragments as well. Methods thatcan be used for the fragmentation processes described above can bephysical or enzymatic. Physical means can encompass any chemical processas well as mechanical shearing and sonication. Enzymatic processes forfragmentation that may find use with the present invention can includebut not be limited to endoucleases such as S1 nuclease, mung beannuclease, RNase, DNase and restriction enzymes. It is a further point ofthe present invention that analytes can be treated with phosphatases ifrequired, to provide an extendable 3′ end. The CNAC of the presentinvention can comprise DNA, RNA or any combination thereof and thenucleotides may be modified or unmodified as desired. The CNACs maycomprise standard nucleotides or they may comprise nucleotide analogs,sugar analogs and phophate analogs. Examples of each of these arepeptide nucleic acids (PNAs), arabinosides and phosphorothioatelinkages.

b. CNAC for Site Specific Fragmentation

The utility of universal bases to providing stability without addingcomplexity finds application in other processes as well. Another aspectof the present invention discloses compositions and methods forcontrolled fragmentation of an analyte or library of analytes. A novelCNAC is disclosed that comprises two segments, a first segment thatcomprises universal nucleotides to provide non-specific binding and asecond segment with a discrete selected sequence that will generate acomplex that provides endonucleolytic digestion. Under appropriatehybridization conditions, the CNAC will create an endonucleasesusceptible site at each location in the analyte that is sufficientlycomplementary to the second segment of the CNAC. The size and nature ofthe selected sequence will determine the average spacing betweenendonuclease sites and therefore the particular average size offragments. For example, a CNAC that comprises a second segment with 4 ormore deoxyribonucleotides should form a complex that is a substrate forRNase H. This should lead to a scission at each site in the analyte thatis complementary to the second segment. On average, a given 4 basesequence should appear about every 250 bases. A smaller sizedistribution can also be obtained by the use of more than one CNACthereby increasing the number of potential digestion sites. Ifpreferred, a larger second segment can be used andhybridization/digestion conditions applied such that the complex isformed at more infrequent intervals and hence a larger averagedistribution in fragment sizes. Specificity may also be increased by theaddition of discrete bases in either the first or third segments andusing conditions such that stable hybrids are only formed with stabilitygenerated by proper base pairing of these bases as well.

The same method can also be applied to digestion of single-stranded DNA.The second segment of a CNAC can be designed with a recognition site fora restriction enzyme. Since most restriction sites are only 4 to 6bases, the presence of the universal bases in the CNAC should provide amuch more stable hybrid than using a 4 to 6 base segment alone. Althoughin this particular embodiment of the present invention, the secondsegment is used for fragmentation, it may also be used as a template forstrand extension for incorporation of a desirable nucleic acid sequenceinto a fragmented analyte after endonucleolytic digestion. As describedpreviously, this can provide a means for template-dependnentincorporation of a labeled nucleotide or oligonucleotide to label theanalyte fragments at their terminus.

If preferred, the CNAC disclosed above can further comprise a thirdsegment. For instance, the third segment can comprise another set ofuniversal bases flanking the other side of the discrete bases in thesecond segment. This CNAC could be represented by the formula“U_(n)-D_(p)-U_(q)” where the “n” represents the number of universalbases in the first segment, “p” represents the number of discrete basesin the second segment and “q’ represents the number of universal basesin the third segment. The additional third segment can provideadditional stability or it may make the hybridized second segment a moreefficient enzyme substrate for endonucleolytic digestion. Alternatively,the first and second are as described above and the third segment is adiscrete nucleic acid sequence that provides a template forincorporation of one or more labels or a desirable nucleic acid sequenceas described previously. Since the universal bases allow forindiscriminate binding, the reactions can take place under conditionswhere only hybridization events that include proper alignment with thediscrete bases in a CNAC form stable hybrids between the CNAC and theanalyte. Alternatively, thermocycling can be carried out to dissociateCNACs that are non-productively bound and allow additional bindingevents that lead to site-specific fragmentation until substantially allof the desired sites on the analyte have been digested.

c. CNACs for Digestion/Extension

In another aspect of the present invention, novel CNACs are disclosedthat comprise at least two segments where the first segment iscomplementary to a first analyte nucleic acid sequence and the secondsegment is complementary to a second analyte nucleic acid sequence. TheCNAC is designed such that after mixing it with an analyte nucleic acid,hybridization of a first segment to a first analyte nucleic acidsequence forms a first complex that is resistant to a particularendonuclease, while hybridization of a second segment to a secondanalyte nucleic acid sequence forms a second complex that is a substratefor the endonuclease. Furthermore, the second complex is capable ofasymmetric cleavage such that only the analyte strand is subject tonicking or removal of nucleotides by the endonuclease. This treatmentgenerates a new 3′ end in the analyte strand that can then be used forthe template dependent addition of nucleotides or oligonucleotides tothe analyte strand.

The CNAC may further comprise a third segment that may or may not becomplementary to a third analyte nucleic acid sequence. The thirdsegment of a CNAC is distinguished from a second segment in that a thirdsegment does not generate a third complex that is sensitive toendonucleolytic digestion. When the third segment is not complementaryto the third analyte nucleic acid sequence, a third complex is neverformed. On the other hand, when the third segment is complementary to athird analyte nucleic acid sequence, a third complex is formed, butendonuclease resistance is endowed by any of the means that can beemployed to render a first complex resistant. After endonucleasedigestion, the sequences in the second and third segments may act astemplates for strand extension from a 3′ end that has been generated byaction of the endonuclease. The strand extension may be carried out by atemplate-dependent polymerizing enzyme (DNA polymerase or reversetranscriptase), or a template dependent ligation enzyme (DNA ligase).Fragments generated by endonuclease digestion may be further besubjected to kinase or phosphatase treatment, in order to add or removephosphate groups at the 3′ or 5′ end as may be desired.

Analytes that may find use in the present invention can be either be DNAor RNA depending upon the nature of the CNAC and the endonuclease.Sequences in the analytes that may be used in the present invention maybe discrete individual sequences, consensus sequences, or genericsequences that are present in all or most of a library of analytes.Examples of RNA that may find use with the present invention can includebut not be limited to hnRNA, rRNA, mRNA, tRNA, or snRNA. Examples of DNAthat may find use with the present invention can include but not belimited to chromosomal, single-stranded, plasmid, viral, bacterial DNA.Digestion of the second complex can be carried out by endonucleases suchas RNase H and restriction enzymes. Prior to hybridization with theCNAC, the target nucleic acid or analyte nucleic acid may also haveundergone pre-treatments including, digestion, fragmentation, extractionand separation. These fragmentation pre-treatments can include physicalmeans, such as shearing, sonication or chemical treatment.Pre-treatments may also include endonuclease or exonuclease digestions.Examples of endonucleases that might find use in the present inventionfor pre-treatment can include but not be limited to S1 nuclease, mungbean nuclease, restriction enzymes, DNAse, ribonuclease H and otherRNases.

The various segments of chimeric nucleic acid construct polymer may becomprised of the same or different backbones. For example, a firstsegment of a CNAC can comprise oligo-ribonucleotides and the secondsegment can comprise oligo-deoxyribonucleotides. Generally, thesugar-phosphate backbone may comprise a natural element, such asphosphate, ribose, or deoxyribose, or it may comprise analogs ofphosphates such as phosphorothioates, or analogs of sugars such asarabinosides. If desired, the 3′ or 5′ end of a CNAC may be blocked toprevent it from acting as a primer or from participating in ligation.The segments of a CNAC may further be comprised of a synthetic backbone,such as a polypeptide. Any synthetic polymer can be used as backbone aslong as bases can be added in the proper orientation so that basepairing can take place. A prominent example of such a synthetic polymerthat has this capability and usefulness is a peptide nucleic acid (PNA).The bases may be comprised of natural purine and pyrimidine bases aswell as modified versions thereof. The bases may also comprise analogsof natural bases. For instance, the universal bases discussed previouslymay also find use in this embodiment of the present invention. Differentsegments of a CNAC may comprise the same or different backbones andcomprise any base structures or elements, depending on the desirablefunction. Thus, one can construct a desired CNAC from the variouscomponents and elements provided above. The particular choice ofcomponents will depend upon the nature of the analyte and theendonuclease to be used.

For example, if the analyte is an RNA molecule, RNase H can be used asthe endonuclease when the backbone of the first segment comprises anoligo-ribonucleotide and the backbone of the second segment comprisesoligo-deoxyribonucleotides. Consequently, hybridization of the firstsegment to an RNA analyte creates a double-stranded RNA first complexthat is resistant to ribonucleaseH and an RNA-DNA second complex whichis a substrate for Rnase H activity. Treatment with RNase H wouldasymmetrically cleave all or some of the portion of the RNA analyteinvolved in the second complex but leave the RNA-RNA hybrid of the firstcomplex and the second segment of the CNAC intact. As described above, aCNAC may also comprise a third segment. In the example above, if thethird segment is complementary to the RNA analyte, the third segment mayalso comprise an oligo-ribonucleotide such that hybridization to theanalyte forms an RNA-RNA hybrid that is resistant to the action of RNaseH. Alternatively as described above, the third segment is notcomplementary to the RNA analyte and no hybrid is formed.

The choice of the particular endonuclease used to carry out this aspectof the present invention depends upon a number of factors. The primaryfactor is the nature of the analyte since the endonuclease must be ableto utilize the analyte as a substrate for nicking or removal ofnucleotides. Secondly, the endonuclease must allow circumstances wheresuch nicking or removal is substantially asymmetric and takes place inthe analyte strand. Thirdly, the endonuclease must allow circumstanceswhere a first or third complex can remain substantially resistant to theaction of the endonuclease. Lastly, the endonuclease must havesufficient specificity that it acts only upon the portion of an analytethat participates in formation of a second complex with the CNAC. It canbe seen that the illustrative example with RNase H described abovefulfills all of these criteria.

Another illustrative example would be to utilize an endonuclease thatintrinsically provides an asymmetric cleavage. For example, digestion ofdouble stranded DNA with the restriction enzyme N.BstNB I results in anick in only one strand 4 bases downstream from the recognition sequence5′ GAGTC 3′. Thus, one could design the second segment of a chimericnucleic acid construct with an oligo-deoxyribonucleotide sequence thatis complementary to this sequence and a first segment that iscomplementary to sequences that are adjacent to the binding site for thesecond segment. In such a manner, when a second complex is formed byhybridizing the CNAC to the analyte, the double-stranded DNA is asubstrate for this specific restriction enzyme and only the analytesequence will undergo cleavage. As described previously, the CNAC cancomprise a third segment that can serve as a template for introductionof a novel nucleic acid sequence by addition to the 3′ end of the nickcreated by the endonuclease digestion. This example also serves as anillustration that a CNAC can still be considered “chimeric” even when itis a chemically homogeneous molecule. For instance, the CNAC above canbe synthesized with three segments that comprise onlyoligo-deoxyribonucleic acids. In the present invention, this would stillbe a chimeric molecule since each segment has a different functionalproperty, i.e., the first segment provides complementary base pairingand stability; the second segment provides for endonucleasesusceptibility and the third segment provides a template for strandextension. This method may also be combined with other embodiments ofthe present invention that have been disclosed previously. For instance,a CNAC with two segments can comprise universal bases with specificnucleotides only in the sites that are required for recognition anddigestion by the asymmetric endonuclease described above.

Another illustrative example of how this aspect of the present inventioncould be carried out would be by the use of an artificial or syntheticsecond segment where the constituents are modified or comprise analogs.Any such modification or analog may be used for this purpose as long asa) they allow hybridization to occur between the second segment and theanalyte b) hybridization with the analyte forms a complex that issusceptible to endonuclease digestion and c) the second segment remainssubstantially resistant to the action of the endonuclease. For example,a second segment could comprise phosphorothioate linkages between bases.It has previously been shown that when a restriction enzyme site in adouble stranded molecule comprises an unmodified segment and aphosphorothioate segment, only the unmodified segment undergoes acleavage event (U.S. Pat. Nos. 5,270,184 and 5,455,166; incorporatedherein by reference). Thus a CNAC with one or more phosphorothioatelinkages in a restriction enzyme sequence in a second segment can behybridized to a complementary segment of an analyte and only the analytestrand should be subject to endonuclease digestion.

Generally, the third segment may contain any arbitrary sequence segment,either related or non-related to a target nucleic acid. The thirdsegment provides a template upon which the cleaved target nucleic acidor the analyte can act as a primer and thereby allow the introduction ofany desirable nucleic acid sequence into an analyte. Through such atemplate dependent sequence introduction to an analyte, a signalingmoiety or other elements such as primer binding sequences can beintroduced directly to an analyte. Furthermore, through such a method,universal sequences could be introduced to an analyte nucleic acid thatcould act at a later stage as a template for the introduction of auniversal primer or primer directed promoter system to prepare copies ofthe analytes as described in U.S. Pat. No. 5,891,636 and Rabbani et al.,in U.S. patent application Ser. No. 09/896,897; both of which areincorporated by reference. The fact that nucleic acid fragments can beconverted to such a construct through such a method could provide for aneven amplification of nucleic acid libraries without prejudice to 3′ endsequences. Further, such a sequence or sequences could be used forpriming or capturing events directly or after amplification. Optionally,if endonuclease cleavage does not leave free 3′-OH in the remaininganalyte, then the remaining analyte could be treated with phosphatase sothat a 3′-OH is generated which can facilitate a priming event. Washing,melting or separation steps can be employed when and where desirable.Generally, with a chimeric nucleic acid construct with three sequencesegments, one can introduce at will desired nucleic acid sequences atany location into an analyte nucleic acid sequence, including anypossible internal sequence sites.

The various teachings in the present invention allows introduction ofdesirable specific sequences in a template directed manner into ananalyte and thus empowering the analyte or set of analytes with diverseproperties and capabilities including: acting as a probe; as a template;as a primer. The CNAC and/or an analyte labeled by means of a CNAC canbe directly or indirectly immobilized onto a solid support which mayinclude: tubes, cuvettes, plates, microtiter wells, beads, magneticbeads, and chips. Methods and compositions for carrying out thisparticular embodiment are described in U.S. Pat. Nos. 4,994,373;4,894,325; 5,288,609; and 6,221,581 B1; 5,578,832; and 5,849,480 (all ofwhich are incorporated by reference). This immobilization can take placeeither before or after strand extension and labeling of an analyte. Forinstance, such capabilities could be used in nucleic acid arrayanalysis, in which instead of probing the analyte, the analyte acts as aprimer on a matrix comprising an array of CNACs that can providetemplates for strand extension of diverse analytes. Depending upon theparticular embodiment of the present invention, hybridized analytes maybe extended directly or undergo an endonuclease step prior to extension.One or more labels or signaling moieties could be incorporated directlyor indirectly with such an array to indicate a specific hybridization ofanalytes to a site on the array.

d. CNAC for Partial Removal of Homopolymeric Sequences

Another aspect of the present invention discloses novel compositions andmethods for the partial removal of a homopolymer sequence. Homopolymericsequences are naturally present in poly A messenger RNA and areartificially present in many methods used for cloning. An example of thelatter is poly C and poly G tailing of double-stranded cDNA molecules(Okayama and Berg, 1982 Mol. Cell. Biol. 2; 161). Although the presenceof these homopolymeric tracts provide beneficial effects for universalprimer binding and cloning, only a small segment is usually necessaryand the presence of large segments may actually be problematic. Forinstance, in a transcription template made from a cDNA copy of mRNA,long homopolymeric segments may induce premature terminations.

As such, the present invention discloses a CNAC that comprises twosegments. The first segment is complementary to a chosen homopolymericsequence and is designed such that a complex formed between thehomopolymeric sequence and the first segment forms a first complex thatis resistant to the action of a particular endonuclease. The secondsegment also comprises a sequence complementary to the homopolymericsequence, but forms a second complex that allows endonuclease digestionof the homopolymer. Thus although each of the segments comprisesequences complementary to the same target sequence, they differ in theproperties they will confer after hybridization.

For instance, a CNAC that comprises a first segment made of rU and asecond segment comprised of dT can hybridize to any segment of a polyAtail of mRNA. Digestion with RNase H will only eliminate poly A segmentshybridized to the second segment. The CNAC can be recycled multipletimes either by using thermal cycling or a temperature where thehybridization through a first segment or a segment alone is insufficientfor stable hybridization. For instance, a CNAC that is comprised of 10rU and 10 dT bases would be able to efficiently hybridize to a 20 basepoly A segment at 37° C. Elimination of rA bases in this segment throughRNAse H activity should destabilize the CNAC, enabling it to bind to anew segment. This process should continue until the mRNA molecules havehas less than 20 rA bases left at their 3′ ends. The remaining smallpoly A segment can then be used as a primer binding site by usingappropriate hybridization conditions. If the CNAC or a DNA primercontaining olgo T is used for this purpose, it is preferred that theRNase. H activity used for the digestion be eliminated prior to priming.

The CNAC described above for generating resistant and sensitivecomplexes is meant only to exemplify the present invention and othersizes may be used for first and second segments. For instance, adeoxyribonucleotide segment of four bases have been shown to besufficient for forming complexes that are substrates for Rnase Hactivity. The size of the segments of the CNAC should be designed suchthat there is efficient complex formation prior to endonucleasedigestion and a sufficient portion of the homopolymeric target remainsintact under the condition used for endonuclease digestion.

Furthermore, the CNACs of the present invention can comprise a thirdsegment that may or may not be complementary to the homopolymeric targetsequence. As described previously, if the third segment iscomplementary, the nature of the endonuclease and third segment is suchthat a third complex remains resistant to digestion by the endonuclease.The third segment can be homopolymeric or heteropolymeric depending uponits intended purpose. The nucleotides in the various segments of theCNAC may be comprised of natural bases or analogs thereof, universalbases or combination thereof that may provide either a weaker orstrengthened hybrid formation with a desired sequence. For instance, theuse of universal bases in a third segment can allow synthesis of acomplementary segment that has a weaker than normal binding. Thus, ifthe new segment on the analyte is desired to be used as a primer bindingsite, a primer with normal base that were complementary to the primerbinding site would have a competitive edge over re-annealing by theuniversal bases in the CNAC.

It will be readily appreciated by those skilled in the art that any ofthe compositions, solid supports, reagents, dyes, primers, nucleic acidconstructs, and the like, can be formulated as kits, which can beemployed for carrying out any of the processes described or claimedherein, and variations of such processes. For example, kits can beformulated as protein or nucleic acid labeling kits, nucleic acidprocessing kits, kits for incorporating desired nucleic acid sequences,amplification kits for amplifying targets, analytes and even a libraryof analytes. Post-synthetic and real time amplification kits can also beformulated from the compositions, solid supports, reagents, dyes,primers, nucleic acid constructs, and the like.

The following examples are offered by way of illustration and not by wayof limitation to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Preparation of Cy 3Labeling Reagent (a) Preparation of Compound I(2,3,3-Trimethylindolinium 5-Sulfone)

P-Hydrazinobenzenesulfonic acid (250 g) was mixed with glacial aceticacid (750 ml) and 3-methyl-2-butanone (420 ml) and heated at reflux for3 hr. The solution was poured into a 2 L beaker and allowed to coolovernight. The resultant suspension was filtered, washed with aceticacid and lyophylized to remove residual acetic acid. The resultant solidwas dissolved in methanol (1.5 L) and a saturated solution of potassiumhydroxide in 2-propanol (900 ml) was slowly added. The color of thesolution turned progressively lighter as the potassium salt of2,3,3-trimethylindolinium 5-sulfone precipitated. The precipitate wasfiltered by suction, washed with 2-propanol and lyophilized to drynessto give 238 g of Compound I.

(b). Preparation of Compound II (1-Ethyl-2,3,3-Trimethylindolenineninium5-Sulfone)

A portion (78 g) of Compound I synthesized in step (a) was suspended in1,2-dichlorobenzene (700 ml). Ethyl iodide (250 ml) was added and themixture was heated at 90-100 C for 12 hr while stirring. The mixture waspoured into 3 L of a 1:1 mixture of ethylacetate/ether and stirred for 2hours. The resulting precipitate was filtered, washed with a 1:1 mixtureof ethylacetate/ether and air-dried to give 68 g of product, CompoundII.

(c) Preparation of Compound III (6-Bromohexanoyl Allyl Amide)

6-Bromohexanoic acid (20 g) and N-hydroxysuccinimide (15 g) weredissolved in 200 ml of anhydrous dimethylformamide (DMF).Dicyclohexylcarbiimide (22 g) in anhydrous DMF (50 ml) was added and themixture was left at room temperature overnight. The precipitated ureawas removed by filtration and the DMF solution containing the product,N-hydroxysuccinimide-6-bromohexanoate, was cooled to −10 to −20° C. Anequimolar amount of allylamine in H₂O (11 ml) was first brought to pH8-9 with glacial acetic acid and then added slowly with stirring to theactive ester. Solid sodium bicarbonate (10 g) was added slowly to avoidexcessive foaming and the mixture was left without covering until thetemperature was raised to −10° C. in two hr. The mixture was poured intoH₂O (1 L) and the product was extracted twice with chloroform (300 ml).The extracts were washed once with 1 N HCl in H₂O, once with 5% NaHCO₃(300 ml) and three times with 10% NaCl in water. The chloroform phasewas dried by addition of solid MgSO₄ and leaving it overnight understirring. The chloroform was removed by evaporation under vacuum leavinga liquid that was used without any further purification for the nextstep.

(d) Preparation of Compound IV (Addition of Linker Arm to Compound III)

Compound I (11 g) from step (a) and Compound III (15 g) from step (c)were dissolved together in 1,2-dichlorobenzene (100 ml) and heated at110° C. for 12 hours while stirring under argon. The mixture was slowlypoured into ethylacetate a 1:1 mixture of ethylacetate/ether (700 ml)and after 30 minutes the solid precipitate was filtered, washed with a1:1 mixture of ethylacetate/ether, air-dried and set aside. A glassysolid that was formed at the bottom of the flask was crushed in amortar, triturated with a 1:1 mixture of ethylacetate/ether, filtered,washed with 2-propanol, dried in vacuum and combined with theprecipitate from above to give Compound IV which was used without anyfurther purification.

(e) Synthesis of Cy 3 Labeling Reagent (Compound V)

A portion of Compound II (12 g) from step (b) andN,N′-diphenylformamidine (10 g) in acetic acid (60 ml) were heated at100-110° C. for 90 min with stirring. During the reaction the absorptionat 286 nm and 415 nm was measured. The ratio of 415/286 increased duringthe first 60 minutes then remained constant at 2.2 for the next 20minutes. After 90 minutes, the hot mixture was poured slowly into 700 mlof a 1:1 mixture of ethylacetate/ether. The resultant solid precipitatewas collected with a pressure filter funnel, washed with 1:1 mixture ofethylacetate/ether and dried by passing argon through the cake. Theprecipitate was collected from the pressure filter funnel and slowlyadded to a mixture of 6.5 g of Compound IV from step (d), 50 ml ofpyridine and 50 ml of acetic anhydride. The progress of the reaction wasmonitored by the decrease of absorbance at 385 nm and an increase inabsorbance at 550 nm. The reaction was carried out overnight understirring at room temperature. The absorbance at 550 nm increased withtime followed by a drop in absorbance as the product precipitated out ofsolution. At the end of the reaction, the brown precipitate wascollected and put aside. The liquid portion was treated by the additionof a seven-fold volume of ethylacetate. The precipitate that formed wascollected and combined with the first precipitate. Since pyridine wouldinterfere with a later palladium catalyzed step, any remaining pyridinewas removed by dissolving the combined precipitate in 100 ml of 0.5MTriethylammonium carbonate, pH 8.0 (TEAC). The TEAC was then removed byevaporation under vacuum leaving a solid pellet. This product (CompoundV) was then dissolved in H₂O and kept at −70° C. until ready to be used.

EXAMPLE 2 Preparation of Cy 5 Labeling Reagent (Compound VI)

Compound II (8 g) from step (b) of Example 1 and malonyl aldehyde dianilhydrochloride (10 g) were dissolved in 100 ml of a 1:1 mixture ofglacial acetic acid and acetic anhydride followed by heating at 110° C.for two hours. The mixture was slowly poured into 500 ml of a 1:1mixture of ethylacetate/ether and the precipitate was filtered, washedwith a 1:1 mixture of ethylacetate/ether and dried by argon as above.The precipitate was then slowly added to a mixture of 12 g of CompoundIV dissolved in 150 ml of a 1:1 mixture of pyridine/acetic anhydridewhile stirring. The mixture was transferred to an oil bath maintained at90-100° C. for 30 minutes while continuing to stir. If desired, thisstep could have been extended up to 90 minutes. The reaction mixture wasthen cooled to room temperature and the precipitate was processedfurther as described previously for the Cy 3 labeling reagent in Example1.

EXAMPLE 3 Attachment of Cy 3 (Compound V) to dUTP

Mercurated dUTP (30 moles) prepared as described in U.S. Pat. No.5,449,767 was dissolved in 1 ml of 1M Lithium acetate and the Cy 3labeling reagent (60 mmol, 0.6 ml) prepared in Example 1 (Compound V)was added with stirring. Potassium tetrachloropaladate (30 mmol in 0.5ml H₂O) was added under argon. The reaction was monitored by HPLC andwas complete after 1 hr at 40° C. Overnight incubation did not increasethe yields. Four volumes of acetone were added to the reaction mixtureand left overnight at −20° C. The next day, the precipitate wascollected by centrifugation.

The pellet was dissolved in 0.1M Lithium acetate (pH 4) and loaded ontoa DEAE Sephadex A₂₅ column. The column was developed by passing througha linear gradient of 0.1-0.7 M LiCl in 0.1M Lithium acetate. Thefractions were examined by HPLC and the fractions which contained asingle late peak were collected and set aside. Another group offractions exhibited two peaks: the late peak described above and anearlier peak. These fractions were combined, adjusted to 0.1M LiCl,reloaded onto a DEAE Sephadex A₂₅ column and refractionated as above.Again the fractions containing a single late peak were collected and setaside. Although it was not done in this example, the fractions thatcontained two peaks after the second chromatography could have beencombined and put onto the column another time to increase the yield ofthe single peak product. The fractions that had exhibited a single latepeak by HPLC were combined together and the H₂O was removed byevaporation in vacuum. The last traces of H₂O were removed byresuspension of the semi-solid residue in 50 ml of 100% ethanol followedby evaporation. The alcohol step was repeated once more. The residue wasresuspended in 30 ml of ethanol and 1 ml of 3M lithium acetate wasadded. The solution was mixed well and left overnight at −20° C. tofacilitate complete precipitation of the Triphosphate. The precipitatewas collected by centrifugation, redissolved in H₂O and partiallylyophilized to remove remnants of the ethanol. The amount of product wasmeasured by absorbance at 550 nm and a molar extinction value of150,000. The solution was then readjusted to a stock concentration of 10mM and stored at −70° C.

Although the procedure above describes the preparation of Cy3 labeleddUTP, the same steps could be carried out for the preparation of Cy 5labeled dUTP by the substitution of the Cy5 labeling reagent (CompoundVI from Example 2) instead of the Cy3 labeling reagent (Compound V fromExample 1) used in the example above.

EXAMPLE 4 Preparation of a Labeled Nucleotide with a Rigid Arm Linkerand an Aphenylic TAMRA Analogue (a) Preparation of Compound VII(3,6-Bis-(Dimethylamino)-Xanthene-9-Propionic acid)

3-(Dimethylamino)phenol (5.8 g) was mixed with succinic anhydride (2.1g) and heated at 120° C. for 90 minutes with stirring under argon. Themixture was cooled, H₂O (80 ml) was added and the mixture was heated atreflux for 10 minutes. The water phase was discarded, leaving behind adark brown gummy material. This substance was dissolved by the additionof H₂O followed by an adjustment to pH 10 with 1M NaOH while stirring.The pH of the clear solution was then brought down to 2 by the additionof 1M HCl. The dye was salted out by the addition of NaCl to a finalconcentration of 2.5 M. The precipitate was filtered, washed with NaCl(2.5 M) and dried by lyophilization to give 1.2 g of Compound VII. Thefluorescence spectrum of Compound VII is shown in FIG. 7.

(b) Preparation of Compound VIII (Active Ester of Compound VII)

Compound VII from step (a) was dissolved in chloroform (200 ml) andN-hydroxysuccinimide (1.5 g) was added with stirring. After theN-hydroxysuccinimide went into solution,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2 g) was added and themixture was stirred overnight in the dark. The mixture was extractedwith H₂O (80 mL) and the chloroform phase containing Compound VIII wasdried with anhydrous magnesium sulfate and stored at −20° C.

(c) Preparation of Compound IX (Free Acid Form of Glycerlyglycine)

13 g of glycylglycine was suspended in a mixture of an equimolar amountof triethylamine in 300 ml of anhydrous methanol and mixed with a 1.5molar excess of methyltrifluoro acetic ester. The suspension wasrefluxed until a homogeneous solution was achieved. The methanol wasremoved by rotary evaporation and the residue was suspended in 100 ml ofH₂O. The pH was then adjusted to 10.0 to allow thetrifluoroglycerylglycine to go into solution. The pH was then broughtdown to 1-2 with Hydrochloric acid whereupon the free acid form of thetrifluoroglycerylglycine precipitated out of solution. This mixture wasleft overnight at 4° C. to allow complete precipitation of the product.The next day, the precipitate (Compound IX) was collected by filtrationand then dried

(d) Preparation of Compound X (NHS Ester of Glycerylglycine)

15 g of Compound IX from step (c) was dissolved in 100 ml of DMF and a 2fold molar excess of N-hydroxysuccinimide was added under stirring. A1.1 fold molar excess of dicyclohexylcarbodiimide dissolved in 10 ml ofDMF was then added and the mixture was left overnight at roomtemperature to produce the NHS ester (Compound X).

(e) Preparation of Compound XI (dUTP with Glycylglycine Linker)

10 uMoles of allylamine dUTP were dissolved in 0.5 ml of 0.3M NaHCO₃followed by addition of 15 mmoles of Compound X from step (d) andincubation at room temperature for 2 hours to form Compound XI. LiAc wasthen added to a final concentration of 0.5M and the nucleotide product(Compound XI) was precipitated by addition of 5 volumes of ethanol andleaving the solution overnight at −20° C. The amine was deprotected bydissolving the precipitate in 1M LiOH for 1 hour at room temperature.The solution was neutralized with glacial acetic acid in the cold andthe Triphosphate was precipitated with ethanol as above.

(f) Preparation of Compound XII (dUTP with Tetraglycyl Linker)

Compound XI from step (e) was further treated by repeating step (e) toadd an additional glycylglycine linker unit thereby forming5′-allylamido-(tetraglycyl) amine dUTP (Compound XII). The amine wasdeprotected and the Triphosphate precipitated as described above in step(e).

(g) Preparation of Compound XIII (Attachment of Aphenylic TAMRA Analogueto dUTP)

20 mmoles of Compound XII from step (f) was dissolved in 2 ml of NaHCO₃(0.3 M) and LiCl (0.7 M) and cooled on ice. The active ester of the dye(40 mmoles) in chloroform from step (b) (Compound VIII) was dried invacuum and dissolved in DMF (2 ml). This solution was then added to theice cold dUTP solution and the mixture was stirred in the dark overnightat room-temperature. The mixture was diluted with 20 ml water and loadedonto a DEAE-Sephadex A₂₄ (20 ml) column at 4° C. The column was washedwith TEAC (0.1 M, pH 7.8, 50 ml) and the product was eluted with alinear gradient of 0.1-0.8 M TEAC, pH 7.8. The fractions that were pureby HPLC were combined. The TEAC was removed in vacuum by repeatedevacuations following the addition of water. The residue was dissolvedin lithium acetate (4 M) and precipitated with 4 volumes of absoluteethanol, then dissolved in water and stored at −70 to give 13.6 mg ofCompound XIII.

It should be noted that the example cited above used a tetraglycyl rigidarm linker. The same methods that were described above could have beenused to synthesize compounds with other lengths. For instance, compoundXII (dUTP with tetraglycyl arm) from step (f) could have beenmanipulated further by a repetition of step (e) and adding anotherglycylglycine unit therby creating a hexaglycyl arm. Similarly, theactivation steps described for preparation of glycylglycine (steps c andd) could also been carried out with glycine as the starting materialthereby allowing addition of single glycyl units.

EXAMPLE 5 Preparation of a Labeled Nucleotide with a Rigid Linker Armand an Aphenylic Texas Red Analogue (a) Preparation of Compound XIV(3,6-Bis-Julolidinoxanthen-9-Propionic Acid)

8-Hydroxyjulolidine (10 g) and succinic anhydride (2.6 g) were combinedunder argon and heated at 130° C. for 2 hours with stirring. The mixturewas cooled, H₂O (150 ml) was added and the mixture was refluxed for 15minutes and then cooled. The water layer was discarded and the glassydark brown residue was dissolved by the addition of H₂O followed by anadjustment to pH 10 with 1M NaOH while stirring. The pH of the solutionwas then brought down to 2 by the addition of 1M HCl at which point theproduct precipitated again. The mixture was centrifuged, the supernatantwas discarded and the pellet was washed by suspending it in water andrecentrifuging, The pellet was then lyophilized to give 3.6 g of product(Compound XIV). The fluorescent spectrum of Compound XIV is shown inFIG. 8.

(b) Attachment of Label to a Nucleotide

Subsequent steps for the preparation of the active ester of Compound XIVand attachment to dUTP with a rigid linker arm were carried out asdescribed in Example 4.

EXAMPLE 6 Preparation of Cyanine Dyes with a Rigid Linker Arm (a)Preparation of Compound XV [(2,3,3,Trimethyl-3-H-indol-5-yl) aceticacid]

110 g of 4-Hydrazinobenzoic acid were mixed with 450 ml of glacialacetic acid and 250 ml of 3-methyl-2-butanone under stirring. Themixture was heated at 128° C.-130° C. for 6 hours and left to coolovernight at room temperature. The glacial acetic acid and3-methyl-2-butanone were removed under vacuum and the solid wastriturated with 300 ml H₂O, filtered and washed again with 300 ml H₂O.The cake was subsequently dried under vacuum. The solid was thenrecrystalized from ethyl acetate resulting in Compound XV.

(b) Preparation of Compound XVI (Diglycylallylamine)

Compound X from step (d) of Example 4 was reacted with a 1.2 fold excessof allylamine acetate in a 50:50 mixture of DMF/H₂O. The solution wasmaintained at pH 8 by the addition of triethylamine and the reaction wascarried out for 4 hours at room temperature. The solution was driedunder vacuum and the mixture was triturated with H₂O to removetriethylamine salts. The slurry was filtered, washed with cold H₂O,lyophilized and dried resulting in Compound XVI

(c) Preparation of Compound XVII [(2,3,3Trimethyl-3-H-indol-5yl)acetamido diglycylallylamine]

20.6 g of Compound XV prepared in Example 7 were dissolved in 100 ml ofDMF followed by addition of 20 g of N-hydroxysuccinimide under stirring.A mixture of 22 g of dicyclohexyl carbodiimide dissolved in 30 ml of DMFwas then added. The mixture was left at room temperature overnight andthe next day, urea was removed by filtration. 30 g of Compound XVI fromstep (a) was dissolved in 100 ml of 50:50 mixture of ethanol and 1M LiOHin H₂O to liberate the amine. This solution was neutralized with aceticacid to pH 8 and added to the filtrate above. An equivalent amount oftriethanolamine was slowly added to the solution over a 1 hour period.The mixture was left at room temperature overnight and the resultantprecipitate was filtered and extracted with 500 ml of chloroform toproduce Compound XVII.

(d) Preparation of Compound XVIII [(2,3,3Trimethyl-3-H-indol-5yl)acetamido diglycylallylamido ethylammonium Iodide]

Chloroform was removed from Compound XVII by vacuum. The glassy residuewas dissolved in 200 ml of DMF followed by removal of the DMF by vacuum.The residue was mixed with 150 ml dichlorobenzene and 100 ml ethyliodideand refluxed at 16 hour at 100° C. After cooling, the solvent wasremoved by decantation. The glassy residue was triturated with ether toproduce Compound XVIII.

(e) Preparation of Cyanine Dyes and Cyanine Dye Labeled Nucleotides

Compound XVIII was used without any further purification to synthesizethe cyanine dyes as described in Examples 1 and 2. The structure of a Cy3 analogue made with Compound XVIII is given below.

The presence of the terminal alkene bond allowed labeling of dUTP asdescribed in Example 3. When tested in a conventional cDNA synthesisassay, significantly higher incorporation was seen with dUTP labeledwith Compound XVIII as compared to a commercially available Cy-3 labeleddUTP (Cat. No. PA 530220) from Amersham Biosciences Corp., Piscataway,N.J.

EXAMPLE 7 meta-EthD, with and without DNA a) Synthesis of meta-EthD

The synthesis of meta-EthD was carried out according to the methoddescribed by Kuhlmann et al., supra. A diagram of the synthetic steps isgiven in FIG. 9. In this procedure, the 2-amino-diphenyl-compound (1)was condensed with acid chloride (2) to give the amide (3) which wasthen converted to a cyclic form to give the phenanthridine (4). Thiscompound (4) was hydrolyzed to give the acid (5), converted to the acidchloride (6) and then condensed with 1,5-diamino-pentane to give thehomodimer. The homodimer was methylated to give (7) which was reduced togive the final product (8) meta-EthD whose structure is given in FIG. 2.

b) Spectral Analysis

meta-EthD was excited at 493 nm and gave emission of 1×10⁵ counts/secondat 617 nm (FIG. 10A). When double stranded DNA was added, the emissionincreased to 6×10⁵ (FIG. 10B). In contrast, when excited at a wavelengthof 350 nm, the emission at 600 nm was 2×10⁴ counts/second that increasedto approximately 3.25×10⁶ counts/second upon the addition of DNA (FIG.11).

EXAMPLE 8 Energy Transfer between a Donor Nucleo Tide and an AcceptorNucleotide

The sequence of an amplicon that can be made from an HIV antisenseconstruct is given in FIG. 12. A description of the derivation of thisconstruct is given in Liu et al., (1997) J. Virol 71; 4079-4085. PCR oftarget analytes in a sample can be carried out in the presence of amixture of dUTP labeled with fluorescein as an energy donor and dUTPlabeled with Compound XIII from Example 4 as an energy acceptor usingthe primers shown in FIG. 12. During the course of amplification,nucleic acid strands are synthesized that incorporate each of theselabels. Illumination at a wavelength appropriate for fluoresceinfollowed by detection at a wavelength appropriate for the emission ofCompound XIII should result in signal generation whenever donornucleotides and acceptor nucleotides are in sufficient proximity. Eithersingle strands or double strands could be analyzed for this purpose.

EXAMPLE 9 Energy Transfer Between an Intercalator and an IncorporatedDye

PCR is carried out using the same primers as used in Example 8. However,in this example, the reaction is carried out in the presence of SYBRGreen and a labeled dUTP from Example 7. As incorporation proceeds,double-stranded DNA begins to accumulate that has Compound XVIII labelednucleotides incorporated. As described previously, SYBR Green displaysenhanced fluorescence after binding to double-stranded DNA. Since SYBRGreen maximally emits at 521 nm and Compound XVIII maximally absorbs at550 nm, fluorescence from Compound XVIII should increase as synthesisproceeds due to energy transfer form SYBR green donors to Compound XVIIIas an acceptor thus indicating successful amplification of targetsequences.

EXAMPLE 10 Energy Transfer Between an Intercalator and an IncorporatedDye with Primers that Comprise Quencher Moieties

This example is carried out as describe in Example 9 except that theprimers are labeled with quenchers as follows:

5′ CAU*GATCCGGAU*GGGAGGTG 3′ and 5′ GCACAU*CCGGAU*AGU*AGA 3′where U* are uridine moieties modifed with a non-fluorescent 3-aminoxanthene as described by Singer and Haugland in U.S. Pat. No. 6,323,337that absorb at about 530 nm. PCR is carried out with these primers inthe presence of a labeled dUTP from Example 7 and SYBR Green asdescribed above. Fluorescence from the intercalated SYBR Green can beabsorbed either by Compound XVIII or by the quencher. If Primer-dimersare formed, these comprise only primers and their complements. As suchenergy transfer should most efficiently take place with the quenchersand thereby reduce spurious signal generation from primer-dimersynthesis. On the other hand, amplicons derived from amplfication oftarget sequences have segments where only compound XVIII is insufficient proximity to the SYBR for energy transfer to take place andtarget dependent signals are generated as synthesis proceeds.

EXAMPLE 11 Energy Transfer Between a Probe and an IncorporatedNucleotide

PCR can be carried out with the same primers used in Example 8. In thisreaction mixture, potential donors are supplied in the form of dUTPlabeled with Compound XVIII form Example 7. The reaction mixture alsocontains a DNA probe labeled with Texas Red moieties that can act asenergy acceptors. The probe has the sequence

5′ U^(F)AATGGU^(F)GAGTATCCCU^(F)GCCTAACTCU^(F) 3′where U^(F) indicates a Uridine labeled with Texas Red. The position ofthis probe in the amplicon is shown in FIG. 12. The probe is alsoblocked at the 3′ end such that it is incapable of being extended. Asamplification is carried out, hybridization of the probe to labeledamplicon strands allow energy transfer to take place between CompoundXVIII and Texas Red that should increase as more amplicon strands aregenerated.

EXAMPLE 12 Endonuclease Digestion and Strand Extension Using aHomopolymeric Target as a Substrate

The steps in this example are shown in FIG. 13. A CNAC with threesegments can be synthesized that has the sequence:

5′-UUUUUUUUUUTTTTQQQQQQQQ-3′where U is a uridine ribonucleotide, T is a thymidinedeoxyribonucleotide and Q is an inosine ribonucleotide and the 3′ endhas been modifed to prevent extension. In this example, theribonuicleotides are 2′-O-methyl as described by Shibahara et al.,(1987) Nucl. Acids Res. 15; 4403-4415 and Baranov et al., (1997) Nucl.Acids Res. 25; 2266-2273 (both of which are incorporated by reference).The CNAC can be hybridized to a library of poly A mRNA (step A) forming:

a first complex with the oligo-uridine first segment bound to a portionof the poly A tail,

a second complex with the oligo-thymidine second segment bound to asecond portion of the poly A tail; and

a third complex with the oligo-inosine third segment bound to a thirdportion of the poly A tails.

In this example, the first and third complexes will be resistant to theactions of RNase H and the second complex should form a substrate forRNAse activity since four deoxyribonucleotides are known to besufficient. Digestion with RNase H at 20-25° C. (step B) should inducecleavage in the poly A segment bound to the oligoT's in the secondcomplex and release of the cleaved poly A tail. Provision of dATP, dCTPand Reverse transcriptase allows extension of the 3′ end of the mRNA(step C). Additionally, if these reagents are present during the RNase Hdigestion step they may help stabilize the binding of the CNAC to the 3′end after endonucleolytic cleavage as described previously. It should benoted that although the inosine is capable of binding to the poly Asegment, when it is used as a template, it preferentially incorporatescytosine thereby introducing a new oligo-C segment into the end of themRNA. Removal of the CNAC allows the oligo-C segment to be used asprimer binding site for an oligonucleotide containing a complementaryoligo-G segment and an RNA promoter sequence. Synthesis of a cDNAstrand, production of a second cDNA strand and generation of a labeledlibrary can then be carried out by any method described previouslyincluding U.S. Pat. No. 5,891,636 and Rabbani et al. in U.S. patentapplication Ser. No. 09/896,897 filed Jun. 30, 2001.

EXAMPLE 13 Addition of an RNA Polymerase Sequence to an Analyte

This example is carried out as described in Example 12 except that thethird segment of the CNAC comprises unmodified ribonucleotides andcontains the sequence for an RNA promoter. As such, after strandextension in step (c), a new third complex is formed where the extendednucleotides are deoxyribonucleotides and the CNAC third segmentcomprises ribonucleotide. This is a substrate for RNase H digestionwhich can then be used to generate a single-stranded segment at the 3′end of the mRNA that is complementary to the RNA promoter sequence. Aprimer with promoter sequence can then be hybridized to the extendedsegment of the mRNA to synthesize a cDNA with a promoter at the 5′ end.Subsequent events can be carried out as described in Example 12. Theremaining portion of the CNAC can be removed prior to binding of theprimer or extension of the primer can allow a strand displacement event.

EXAMPLE 14 Preparation of a Dioxetane Derivative that is Capable ofLight Generation after an Enzyme Catalyzed Intrachain Rearrangement

A schematic of the steps that can be used to synthesize an intermediatecompound for derivatization of a dioxetane is shown in FIG. 14. Theseries of steps shown in this schematic can be carried out usingstandard chemistry methods. In the last step of this procedure, compound(e) can be attached to a dioxetane derivative where both “Q” and “Z” areas described previously. This dioxetane derivative (f) comprises an R1and an R2 group joined to adjacent sites of a cyclic ring as disclosedand defined in the present invention.

EXAMPLE 15 Potential Series of Enzyme Dependent Events with Compound (f)from Example 14

In the presence of Acylase I, a cleavage event takes place thatgenerates a free primary amine as shown by compound (f) being convertedto compound (g) in FIG. 15. Due to the proximity of the released primaryamine to the benzoyl residue in compound (g) and subsequent formation ofa six-membered ring in the transition state, compound (h), the internalrearrangement to produce compound (i) is a very rapid reaction. Thepresence of the phenoxy group in compound (i) makes it an unstabledioxetane that should generate light as it decomposes. It has beenpreviously described in the literature that a similar displacement cantake place with an acyl residue and a primary amine or thiol at the endof a chain attached to the acyl group. These previously describedreactions should not have the favorable kinetics of the reaction shownin this example.

This example shows an enzymatic reaction that converts R1 into R1*thereby producing a reactive group G1 that is at the end of a chainattached to one site of a cyclic ring. In this particular example,Acylase I is the enzyme and G1 is a free primary amine. The reactioncontinues with G1 interacting with a benzoyl group (G2) that is attachedto a different site on the cyclic ring. This intermediate is shown as(h) in FIG. 15. An internal rearrangement takes place between the amineand benzoyl group (G1 and G2 respectively) leading to the intrachaintransfer of the benzoyl group and generation of an unstable lightemitting dioxetane.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art in light of the above detailed description and examplesof the present invention. All such variations are fully embraced by thescope and spirit of the invention as more particularly defined in theclaims that now follow.

1. A labeling reagent comprising a nonmetallic porphyrin, said reagentcomprising:

wherein R₀ is a reactive group and is attached directly or indirectly toany one of the four non-pyrrole positions of said nonmetallic porphyrin,and R₁ through R₈ independently comprise hydrogen, aliphatic,unsaturated aliphatic, cyclic, heterocyclic, aromatic, heteroaromatic,charged or polar groups, or any combinations of the foregoing, whereinsaid reactive group R₀ comprises sulfhydryl, hydroxyl, amine,isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituteddiazine, maleimide, aziridine, sulfonylhalide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-proprionamide,glyoxal, an alkene group, an alkyne group or a halogenated compound. 2.The labeling reagent of claim 1, wherein any of said R₁ through R₈ alkylgroups comprises methyl, ethyl or propyl.
 3. The labeling reagent ofclaim 1, wherein any of said R₁ through R₈ alkyl groups furthercomprises a polar or charged group.
 4. A labeling reagent comprising anonmetallic porphyrin, said reagent comprising:

wherein R₀ is a reactive group and is attached directly or indirectly toany one of the four non-pyrrole positions of said nonmetallic porphyrin,and R₁ through R₈ independently comprise hydrogen, aliphatic,unsaturated aliphatic, cyclic, heterocyclic, aromatic, heteroaromatic,charged or polar groups, or any combinations of the foregoing, whereinsaid reactive group R₀ comprises sulfhydryl, hydroxyl, amine,isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituteddiazine, maleimide, aziridine, sulfonylhalide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-proprionamide,glyoxal, aldehyde, or a moiety capable of forming a carbon-carbonlinkage with a target wherein said moiety is an alkene group, an alkynegroup or a halogenated compound, and wherein said reactive group R₀ isattached indirectly to said nonmetallic porphyrin through a linker arm.5. A labeling reagent comprising a nonmetallic porphyrin, said reagentcomprising:

wherein R₀ is a reactive group and is attached directly or indirectly toany one of the four non-pyrrole positions of said nonmetallic porphyrin,and R₁ through R₈ independently comprise hydrogen, aliphatic,unsaturated aliphatic, cyclic, heterocyclic, aromatic, heteroaromatic,charged or polar groups, or any combinations of the foregoing, whereinsaid reactive group R₀ comprises sulfhydryl, hydroxyl, amine,isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituteddiazine, maleimide, aziridine, sulfonylhalide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester,hydrazine, azidonitrophenyl, azide, 3 -(2-pyridyldithio)-proprionamide,glyoxal, aldehyde, or a moiety capable of forming a carbon-carbonlinkage with a target wherein said moiety is an alkene group, an alkynegroup or a halogenated compound, wherein said reactive group R₀ isattached indirectly to said non-metallic porphyrin through a linker armand said linker arm comprises at least two consecutive peptide bonds. 6.A labeled target comprising a nonmetallic porphyrin, said reagentcomprising:

wherein T is a target molecule attached directly or indirectly to anyone of the four non-pyrrole positions of said nonmetallic porphyrin andR₁ through R₈ independently comprise hydrogen, aliphatic, unsaturatedaliphatic, cyclic, heterocyclic, aromatic, heteroaromatic, charged orpolar groups, or any combinations of the foregoing, wherein said targetT comprises a nucleic acid, a nucleotide or a nucleic analog, areceptor, a hormone, a lymphokine, a cytokine, a toxin, a carbohydrate,a sugar or an oligo- or polysaccharide.
 7. The labeled target of claim6, wherein said nucleic acid or nucleotide or nucleotide analog ismodified.
 8. The labeling reagent of claim 6, wherein any of said R₁through R₈ alkyl groups comprises methyl, ethyl or propyl.
 9. Thelabeling reagent of claim 6, wherein any of said R₁ through R₈ alkylgroups further comprises a polar or charged group.
 10. The labelingreagent of claim 6, wherein said target T is attached indirectly to saidnonmetallic porphyrin through a linker arm.
 11. The labeling reagent ofclaim 10, wherein said linker arm comprises at least two consecutivepeptide bonds.